EP2467470A2 - Cellules progénitrices de péricyte et leurs procédés de génération et d'utilisation - Google Patents

Cellules progénitrices de péricyte et leurs procédés de génération et d'utilisation

Info

Publication number
EP2467470A2
EP2467470A2 EP10755000A EP10755000A EP2467470A2 EP 2467470 A2 EP2467470 A2 EP 2467470A2 EP 10755000 A EP10755000 A EP 10755000A EP 10755000 A EP10755000 A EP 10755000A EP 2467470 A2 EP2467470 A2 EP 2467470A2
Authority
EP
European Patent Office
Prior art keywords
cells
pericyte
cell
isolated
isolating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10755000A
Other languages
German (de)
English (en)
Inventor
Ayelet Dar-Oaknin
Joseph Itskovitz-Eldor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technion Research and Development Foundation Ltd
Original Assignee
Technion Research and Development Foundation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technion Research and Development Foundation Ltd filed Critical Technion Research and Development Foundation Ltd
Publication of EP2467470A2 publication Critical patent/EP2467470A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention in some embodiments thereof, relates to isolated pericyte and endothelial progenitor cells from pluripotent stem cells, and methods of generating and using same.
  • microvessels including capillaries, precapillary arterioles, postcapillary venues and collecting venules are composed of internal endothelial layer surrounded by outer coverage of pericytes (aka Rouget cells or mural cells), which are also called mesangial cells in the kidney and Ito cells in the liver.
  • pericytes aka Rouget cells or mural cells
  • perivascular SMC and pericytes have been shown to function as critical regulators of vascular development, stabilization, maturation, and remodeling mediated by TGF- ⁇ , PDGF-B, SPl or Angl [Armulik et al., 2005, Circulation Research, 97: 512-523; Bergers and Song, 2005, Neuro-Oncology, 2005, 7: 452-464)].
  • pericytes Although related in function and anatomical localization pericytes can be distinguished from SMC based on their characteristic morphology, and specific cell marker expression. Thus, while SMC form a separate layer of the tunica media in blood vessels, pericytes are physically embedded within the endothelial basement membrane to promote mutual communication with the underlying endothelium. In addition, SMC and the majority of pericytes in m ⁇ ltiple human and murine tissue types express ⁇ - smooth muscle actin ( ⁇ -SMA), which is involved in regulation of vessel contractility.
  • ⁇ -SMA ⁇ - smooth muscle actin
  • Crisan M., et al. describe isolation and culturing of human perivascular cells (pericytes) from various adult and fetal tissues which were shown capable of differentiating into myogenic, osteogenic, adipogenic and chondrogenic cell in vitro.
  • the cultured pericytes stably expressed NG2, CD146, ⁇ - SMA, PDGF-R ⁇ and alkaline phosphatase but not markers of endothelial cells (CD34, CD 144, CD31, and vWF), hematopoietic cells (CD45) or myogenic cells (myogenin, m- cadherin, myf-5 and Pax 7).
  • hESC human embryonic stem cells
  • iPSC human induced pluripotent stem cells
  • Levenberg S., et al. (PNAS USA 2002, 99:4391-96) describe selection of endothelial cells from human embryoid bodies by cell sorting (FACS) using monoclonal antibodies raised against the endothelial-specific marker PECAM-I.
  • FACS cell sorting
  • the selected, PECAM-1+ embryoid body-derived (EBD) cells exhibited endothelial-specific characteristics such as von Willebrand factor, VEGFR-2 and VE-cadherin surface markers.
  • WO03/087296 and U.S. 7,354,763 disclose a method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells which differentiate into vascular smooth muscle cells.
  • a method of isolating a pericyte progenitor cell from embryoid bodies comprising:
  • a method of isolating a pericyte progenitor cell from pluripotent stem cells comprising:
  • a method of isolating an endothelial progenitor cell from embryoid bodies comprising:
  • a method of isolating an endothelial progenitor cell from pluripotent stem cells comprising:
  • the method further comprising: culturing the CD105+, CD73+ and/or CD105+/CD73+ cells for about one or two passages prior to the isolating the CD31+/UEA-l+/Ve-cadherin+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • a method of co-derivation of pericyte and endothelial progenitor cells comprising:
  • a method of co-derivation of pericyte and endothelial progenitor cells comprising:
  • CD73+ and/or CD105+/CD73+ cells to thereby isolate the endothelial progenitor cells;
  • the method further comprising: culturing the CD31+/UEA-l+/Ve-cadherin+ cells, to thereby expand the endothelial progenitor cells.
  • the method further comprising: culturing the CD31- cells, to thereby expand the pericyte progenitor cells.
  • an isolated pericyte progenitor cell generated according to the method of some embodiments of the invention.
  • an isolated pericyte progenitor cell having a CD105+/CD31-/ ⁇ SMA-/CD133- /FIk-I-, a CD73+/CD31-/ ⁇ SMA-/CD133-/Flk-l- or a CD105+/CD73+CD31-/ ⁇ SMA- /CD133-/Flk-1- signature.
  • an isolated population of cells which comprises at least 85% of the isolated pericyte progenitor cells of some embodiments of the invention.
  • an isolated population of cells comprising at least 85% of pericyte progenitor cells, the pericyte progenitor cells having a CD105+/CD31-/ ⁇ SMA-, a CD73+/CD31- / ⁇ SMA- or a CD105+/CD73+/CD31-/ ⁇ SMA- signature.
  • a cell culture comprising a culture medium and the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention.
  • a method of generating osteoblast cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a culture medium which comprises ⁇ -glycerol-phosphate, Dexamethasone and ascorbic acid, thereby generating the osteoblast cells.
  • a method of generating adipocyte cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a culture medium which comprises IBMX (3-isobutyl-l-methylxanthine), Dexamethasone and insulin, thereby generating the adipocyte cells.
  • a method of generating chondrocyte cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a' culture medium which comprises dexamethasone, ascorbic acid and TGF ⁇ 3, thereby generating the chondrocyte cells.
  • a method of generating myoblast cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a culture medium which comprises horse serum, thereby generating the myoblast cells.
  • a method of generating smooth muscle cells in vivo comprising implanting the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a subject in need thereof, thereby generating the smooth muscle cells in vivo.
  • the isolated pericyte progenitor cells are mixed with an extracellular matrix.
  • an isolated population of pericyte and endothelial progenitor cells generated according to the method of some embodiments of the invention.
  • an isolated population of endothelial progenitor cells generated according to the method of some embodiments of the invention.
  • a cell culture comprising a medium and the isolated population of cells or some embodiments of the invention.
  • a pharmaceutical composition comprising the isolated pericyte progenitor cell of some embodiments of the invention, the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, and/or the isolated population of endothelial progenitor cells of some embodiments of the invention, and a therapeutically acceptable carrier.
  • a method of treating a pathology requiring vascular tissue regeneration and/or repair comprising administering to a subject having the pathology the isolated pericyte progenitor cell of some embodiments of the invention, the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, and/or the isolated population of endothelial progenitor cells of some embodiments of the invention, or the pharmaceutical composition of some embodiments of the invention, thereby treating the pathology.
  • the method further comprising passaging the CD105+, CD73+ and/or CD105+/CD73+ cells for at least 2 passages to thereby expand a population of pericyte progenitor cells.
  • the method further comprising enriching the cells for CD105+/CD31-, CD73+/CD31- and/or CD105+/CD73+/CD31- cells.
  • enriching is effected by depleting CD31+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the pericyte progenitor cell having a CD105+/CD73+/CD31-/ ⁇ SMA- signature. According to some embodiments of the invention, the pericyte progenitor cell having a CD105+/CD73+/CD31-/ ⁇ SMA-/CD133- signature.
  • the pericyte progenitor cell having a CDIOSH-ZCDTSVCDSl-ZaSMA-ZFIkI- or CD105+ZCD73+ZCD31-ZaSMA- ZCD133-ZFM- signature.
  • the pericyte progenitor cell having a CD105+ZCD73+ZCD31-Z ⁇ SMA-ZNG2+, CD105+ZCD73+ZCD31-Z ⁇ SMA- ZCD133-ZNG2+ or CD105+ZCD73+ZCD31-Z ⁇ SMA-ZCD133-ZFlkl-ZNG2+ signature.
  • the pericyte progenitor cell is CD146+.
  • the pericyte progenitor cell is CD90+.
  • the pericyte progenitor cell is Tie-l+ZTie-2+.
  • the isolated pericyte progenitor cell is capable of differentiation into at least two cell lineages of the cell lineages selected from the group consisting of osteoblasts, chondrocytes, myobloasts and apipocytes.
  • isolating the CD105+, CD73+ andZor CD105+ZCD73+ cells from the embryoid bodies is performed between about day 4 to about day 26 of differentiation of the embryoid bodies.
  • isolating the CD105+, CD73+ andZor CD105+ZCD73+ cells from the embryoid bodies is performed by cell sorting or using magnetic beads.
  • the pericyte progenitor cell exhibits a CD105+ZCD73+ZCD31-Z ⁇ SMA- signature at any passage in culture from passage 1 to senescence.
  • the pericyte progenitor cell exhibits a CD105+ZCD73+ZCD31-Z ⁇ SMA-ZCD133-ZFlk-ZNG2+ZCD146+ZCD90+ZTie- l+/Tie-2+ signature at any passage in culture from passage 1 to senescence.
  • the differentiation into at least two cell lineages is maintained at any passage in culture from passage 1 to senescence.
  • the pericyte being substantially free of contact inhibition when cultured in a two dimensional culture dish.
  • the pericyte adopts a hill and valley morphology when cultured in a two dimensional culture dish.
  • the culturing comprises passaging the pericyte progenitor cell every 3-8 days.
  • the culturing comprises about 7-9 passages.
  • the osteoblast cells are characterized by mineral deposits and massive calcium content.
  • the adipocyte cells are characterized by accumulation of lipid-rich vacuoles which are positive for Oil red staining.
  • the chondrocyte cells are characterized by positive von-Kossa staining.
  • the enriching the cells is effected prior to the culturing the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the pluripotent stem cells are embryonic stem cells.
  • the pluripotent stem cells induced pluripotent stem cells iPS
  • the embryoid bodies are human embryoid bodies.
  • isolating the CD105+ and/or the CD105+/CD73+ cells is effected using an anti CD105 antibody.
  • isolating the CD73+ and/or CD105+/CD73+ cells is effected using a CD73 antibody.
  • isolating the CD105+, CD73+ and/or CD105+/CD73+ cells from the embryoid bodies is performed between about day 7 to about day 26 of differentiation of the embryoid bodies.
  • Implementation of the method and/or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.
  • a data processor such as a computing platform for executing a plurality of instructions.
  • the data processor includes a volatile memory for storing instructions and/or data and/or a non-volatile storage, for example, a magnetic hard-disk and/or removable media, for storing instructions and/or data.
  • a network connection is provided as well.
  • a display and/or a user input device such as a keyboard or mouse are optionally provided as well.
  • FIGs. IA-B depict RT-PCR analyses of vasculogenic markers on developing human PSC-derived EBs.
  • Figure IA - CD105 expression Figure IB - CD31 expression.
  • Kinetic of vascular and perivascular gene expression was detected by RT- PCR from day 1 to day 26 of EBs differentiation in EBs which spontaneously differentiated from pluripotent stem cells: H9.2 (human ESC line) and induced pluripotent stem cells (C3).
  • FIGs. 2A-P are dot plots depicting flow cytometry analyses showing the expression of CD105 and CD31 cells in the developing EBs.
  • EBs were spontaneously formed (differentiated) from pluripotent stem cells: H9.2 (human ESC line; Figures 2A, 2C, 2E, 2G, 21, 2K, 2M and 20) and induced pluripotent stem cells (C3; Figures 2B, 2D, 2F, 2H, 2J, 2L, 2N and 2P) and FACS analysis was performed at the indicated days of EBs differentiation: Day 1 ( Figures 2A-B), day 4 ( Figures 2C-D), day 7 (Figures 2E- F), day 10 ( Figures 2G-H), day 14 ( Figures 21- J), day 19 ( Figure 2K-L), day 21 ( Figures 2M-N) and day 26 ( Figure 20-P).
  • the Y axis in each panel represents CD 105 expression in the dissociated EBs single cells
  • FIGs. 3A-D are histograms depicting percentages of the subpopulations of cells in cells of differentiated EBs. Results are representative to 4 independent experiments using H9.2 and 3 independent experiments using C3 pluripotent stem cells.
  • Figures 3A and 3C White bars: CD31+/CD73-; black bars: CD31+/CD73+; and shaded (grey) bars: CD31-/CD73+.
  • Figures 3B and 3D White bars: CD105+/CD31+; black bars: CD105+/CD31-; and shaded (grey) bars: CD105-/CD31+.
  • the results show the time course of emergence of CD31+CD105+/- endothelial population and non-endothelial CD105+CD31- subset during the onset of vasculogenesis in developing EBs.
  • FIGs. 4A-C are dot plots depicting flow cytometry analyses showing the emergence of pericytes in differentiated PSC-derived EBs. Co-expression of CD 105 and CD90 on a CD31 negative subpopulation of 19 days old 16 hESC-derived EBs. Subsets of CD105+CD90-CD31- and CD105+CD90+CD31- can be identified by flow cytometry analysis.
  • FIGs. 5A-H are fluorescent microscopy images depicting immunofluorescence analyses of differentiating EBs using antibodies specific to the CD31 and CD90 cell surface markers.
  • FIGs. 6A-H are fluorescent microscopy images depicting immunofluorescence analyses of differentiating EBs using antibodies specific to the Calopnin and CD90 cell surface markers.
  • FIGs. 7A-I are microscopy images depicting characterization of PSC-derived colonies generated from isolated CD 105+ single cells at day 7 ( Figures 7 A, 7D and 7G) and day 20 ( Figures 7B, 7C, 7E, 7F, 7H and 71).
  • CD105+ cells were sorted from 14 days old EBs and sparsely-plated (3 cells/cm 2 ) in M- 199 ECs growth medium. Shown are phase microscopy images ( Figures 7A, 7B, 7D, 7E, 7G and 7H) and fluorescence microscopy images ( Figures 7C, 7F and 71).
  • FIG. 9 is a histogram depicting fold increase (expansion) in PSC-derived pericyte number (from both hESC H9.2 and human iPS C3) throughout 60 days of culture expansion (up to 8 passages).
  • FIGs. 10A-P are dot plots depicting flow cytometry analyses of cultured PSC- derived pericytes between passage 1-8, until senescence.
  • the PSC-derived pericytes were isolated from EBs generated by spontaneous differentiation of hESC9.2 ( Figures 1OA, 1OC, 1OE, 1OG, 101, 10K, 1OM and 10O) or hiPSC C3 cells ( Figures 1OB, 10D, 1OF, 1OH, 10J, 1OL, ION and 10P).
  • the cells subjected to analysis were all CD31- (negative for expression of CD31), taken from any passage from the range of passages 1-8.
  • FIG. 11 is a gel image depicting RT-PCR analysis of cultured PSC-derived pericytes between passage 1-8, until senescence.
  • the PSC-derived pericytes were isolated from EBs generated by spontaneous differentiation of hESC9.2 or hiPSC C3 cells. Lanes from left to right: (1) DNA marker; (2) CtI (control); (3) ⁇ SMA; (4) Fibroblast specific protein-1 (fspl) ; (5) CD73; (6) CD90; (7) CD105; (8) Calponin; Note expression of CD73, CD90, CD 105 and Calponin. Consistently, throughout the whole culture, the majority of pericytes were negative for ⁇ -SMA (arrow) and only a small subset of pericytes (about 6%) was positive for ⁇ -SMA.
  • FIGs. 12A-F are fluorescence microscopy images depicting immunofluorescence analysis of cultured PSC-derived pericytes between passage 1-8, until senescence.
  • the PSC-derived pericytes were isolated from EBs generated by spontaneous differentiation of
  • Figures 12A-B double staining of calponin (red) and PDGFR- ⁇ (green).
  • Figures 12C-D - alpha-SMA (red) and NG2 (green).
  • Figures 12E-F CD90 (red) and CXCR4 (green). DAPI (4',6-diamidino-2-phenylindole) stained nuclei in blue.
  • FIG. 13 is a dot plot depicting flow cytometry of the isolated pericyte cells in culture (from passage 1-9 or senescence) obtained according to the method of some embodiments of the invention using alpha smooth muscle actin ( ⁇ SMA) antibodies (clone 1A4, DAKO). Note that while the majority of the cells are ⁇ SMA- (i.e., do not express ⁇ SMA), a small subset of the cells (up to 6%) express ⁇ SMA. Thus, the isolated population of cells includes at least 94% of ⁇ SMA- cells.
  • ⁇ SMA alpha smooth muscle actin
  • FIGs. 14A-B are fluorescence microscopy images depicting immunofluorescence using anti-Tie-1 ( Figure 14A, red) and anti-Tie-2 ( Figure 14B, green) antibodies. Nuclei were stained using DAPI. Tie-1+ (positive for Tie-1) cells are stained in red (with purple nuclei due to DAPI stain. Tie-2+ (positive for Tie-2) cells are stained in green (with blue nuclei due to DAPI stain). Note that all pericyte progenitor cells are Tie-l+/Tie-2+.
  • FIGs. 15 A-B are light microscopy images depicting cord networks formation on Matrigel by PSC-derived pericytes at passage 6 ( Figure 15A) or EPCs ( Figure 15B). Pericytes rearrange in smaller tube-like structures in comparison to those created by EPCs. These results demonstrate that the pericyte cells generated according to some embodiments of the invention are capable of forming vasculogenic structures in vitro.
  • FIGs. 16A-H are microscopic images depicting immunofluorescence analysis of vasculogenic cells assembling into tubular network. Seeding a mixture of PSC-derived endothelial cells identified by immuno-labeling of CD31 (red, Figure 16A and 16E) and carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled PSC-derived pericytes (green, Figures 16B and 16F) on Matrigel result in efficient assembly of the vasculogenic cells into tubular network (merged, Figures 16D and 16H). Nuclear staining by DAPI (Blue, Figures ] 6c and 16G). Original magnifications: x40, Figures 16A, 16B, 16C and 16D xlOO, Figures 16E, 16F, 16G and 16H.
  • FIGs. 17A-E are light microscopy images depicting hematoxylin and eosine (H&E) stains stained sections of implants generated by the pericytes cells and/or endothelial cells of some embodiments of the invention.
  • H&E hematoxylin and eosine
  • Figure 17A empty implant (control Matrigel implant); Figure 17B - Matrigel mixed with PSC-derived EPC only (H9.2) (H9.2-derived EC); Figure 17C - Matrigel mixed with PSC-derived pericytes only (H9.2-derived pericytes); Figure 17D - Matrigel mixed with PSC-derived pericytes mixed with human umbilical vein endothelial cells (H9.2-derived pericytes + HUVECs); Figure 17E - Matrigel mixed with PSC-derived pericytes and endothelial cells (both derived from C3) (iPSC C3-derived ECs and pericytes).
  • H&E staining revealed the presence of luminal structures containing erythrocytes (red, un-nucleated cells) in implants where both vasculogenic cells types (endothelial and pericytes) were used ( Figures 17B and 17C, but not in implants where endothelial cells or pericytes were used alone. Implants of pericytes only induced infiltration of very few murine blood vessel into the Matrigel ( Figure 17C).
  • FIGs. 18A-E are fluorescence microscopy images depicting immunofluorescence analysis of implants generated the pericytes cells and/or endothelial cells of some embodiments of the invention.
  • a total of 1.5xlO 6 pericytes (8xl0 5 -10 6 ) and EPC (5-7xlO 5 ) were re-suspended in 250 ⁇ l Matrigel without adding vasculogenic growth factors or serum to the mixture.
  • Matrigel mixtures were then implanted subcutaneously into immunodeficient 6-8 weeks old NOD/SCID mice, harvested after 7 days, sectioned and immunolabeled with antibodies specific for human over mouse vasculogenic markers.
  • Figure 18A Matrigel mixed with iPSC-derived pericytes.
  • MHC class I red
  • nuclei DAPI
  • Figure 18B Matrigel mixed with PSC-derived pericytes mixed with human umbilical vein endothelial cells (H9.2-derived pericytes + HUVECs).
  • CD31 red
  • MHC class I green
  • nuclei DAPI
  • Figure 18C Matrigel mixed with PSC-derived pericytes and endothelial cells (C3) (iPSC- derived ECs and pericytes).
  • CD34 red
  • MHC class I green
  • nuclei DAPI
  • Figure 18D Matrigel mixed with H9.2-derived endothelial cells and pericytes.
  • CD31 (red), vW factor (green), Nuclei (DAPI, blue); Figure 18E - Matrigel mixed with PSC- derived pericytes and endothelial cells (C3) (iPSC-derived ECs and pericytes) CD31 (red), MHC class I (green), Nuclei (DAPI, blue); Cultured pericytes, expressing the human MHC Class I antigen (red, Figure 18A) mediated formation of vasculogenic structures in vivo within 1 week.
  • Luminal implanted endothelial cells (HUVECs, H9.2 or C3 derived endothelial cells) are immunoreactive for the specific antibody against human CD31 or CD34 endothelial cell marker.
  • FIGs. 19A-F are microscopy images depicting osteogenic characteristics of cultured PSC-derived pericytes in vitro and in vivo.
  • Figures 19A-B Cultured PSC- derived pericytes were stimulated with osteogenic medium for 2-4 weeks. Mineral deposits appear in black (Figure 19A). Robust Alizarin red stained calcium deposits can be seen within 2 weeks in osteogenic medium ( Figure 19B).
  • Figure 19B-inset monolayer of alizarin red stained differentiated PSC-derived pericytes in culture dish. Uniform osteogenic potential was identical throughout the whole culture period from passage 2 to passage 8 until cell senescence, indicating that osteogenic potential of the expanded pericytes was fully maintained.
  • FIG. 19A Figure 19A, xlOO; Figure 19B, xl60.
  • Figures 19C-F Cultured pericytes were cultivated in osteogenic medium for 3 ( Figures 19C-D) or 14 days ( Figures 19E-F), removed, mixed with Matrigel, and implanted subcutaneously in the back of SCID-NOD mice.
  • H&E Figure 19C and Figure 19E
  • Figures 19D and 19F - Alizarin red staining reveals moderate ( Figure 19D, 3 days in osteogenic medium) to massive (Figure 19F, 14 days in osteogenic medium) appearance of calcium deposits corresponding to of length incubation periods with osteogenic medium of implanted pericytes.
  • FIGs. 20A-B are microscopy images depicting adipogenic potential of PSC- derived pericytes in vitro.
  • Cultured PSC-derived pericytes (passages 2-8) were stimulated with adipogenic medium up to 14 days fixed and stained with Oil red O for detection of lipids.
  • H9.2-derived Figure 2OA, xlOO
  • C3 derived Figure 2OB, x200 differentiated pericytes.
  • FIGs. 21A-J are flow cytometry analyses depicting VEGFRl/Flt-1, VEGFR2/Flk-1, CD31 and UEA-I expression pattern of cultured endothelial cells (ECs; Figures 21B, 21D, 21F, 21H, 21J) and progenitor pericytes ( Figures 21A, 21C, 21E, 21G, 211) from pluripotent stem cells (PSCs).
  • PSCs pluripotent stem cells
  • the analyses are representative for passages 1-5 for ECs and 1-8 for pericytes.
  • FIGs. 22A-C depict the potential usage of PSC-derived vasculogenic endothelial cells and pericytes in the clinic.
  • Figure 22A photograph of a critical limb ischemia due to diabetes.
  • Figures 22B-C schematic illustrations of arteriosclerosis.
  • FIGs. 23A-E are schematic diagrams illustrating co-derivation of vasculogenic endothelial and pericytes from human PSC-derived embryoid bodies. Embryoid bodies are spontaneously formed in differentiating medium up to 26 days, 3D differentiating embryoid bodies in feeder free culture ( Figure 23A). Step I: Enzymatic dissociation of EBs with Collagenase B and DNase I results in single cells, of which CD105+ are isolated or sorted for further culture (endothelial and pericyte progenitor cells). Co- cultivation of CD105+ endothelial and pericyte progenitor cells at passage 0 results in 2 cell types: vasculogenic pericytes and endothelial cells ( Figure 23B).
  • step 2 Further culturing results in differentiation of pericyte progenitor cells into vasculogenic pericytes.
  • a second isolation step (step 2) based on CD31+ gives rise to two distinguished population: CD31+UEA-1+CD105+ endothelial cells ( Figure 23D, positive fraction) and CD31-UEA-1-CD105+ pericytes ( Figure 23E, negative fraction).
  • FIGs. 24A-C are dot plots depicting flow cytometry analyses of CD105+ population in human ESC-derived EBs. Dissociated EBs (7 days old) were labeled with either PE-conjugated anti-CD105 (Figure 24A, 3.6% CD31+ of total dissociated EBs), FITC-conjugated anti-CD31 ( Figure 24B, 3.2% CD31+ of total dissociated EBs) or combination of both ( Figure 24C). Subpopulations of CD105+CD31+ (3.1%), CD105+CD31- (0.4%) and CD105-/lowCD31+ (0.1%) can be seen.
  • FIGs. 25A-C are dot plots depicting flow cytometry analyses of CD31+ endothelial progenitor cells, demonstrating enhancement of the percentage of CD31+ endothelial cells after EB-dissociation with Collagenase B and DNase I. Representative percentages of CD31+ populations, from 14 days old dissociated H9.2-derived EBs ( Figures 25 A and 25B). Cells were isolated by magnetic separation using anti-CD31 conjugated beads to yield at least 95% pure CD31+ cell population ( Figure 25C).
  • FIGs. 26A-E depict derivation of two distinguished vasculogenic populations from isolated cultured CD105+ cells at first passage.
  • Figure 26A dot plot of flow cytometry analysis of CD105 + isolated cells using CD31 and VEGFR-PE antibodies
  • Figure 26B Immunofluorescence analysis of CD105+/CD31- or CD105+/UEA-1- using antibodies which stain NG2 (green) and Calponin (red), nuclei are stained with DAPI (blue)
  • Figure 26C Immunofluorescence analysis of CD105+/CD31+ or CD105+/UEA-1+ cells using antibodies which stain vW factor (green) and CD31 (red), nuclei are stained with DAPI (blue)
  • Figure 26D dot plot depicting flow cytometry analysis using CD31 and CD105 antibodies
  • Figure 26E dot plot depicting flow cytometry analysis using UEA-I and VE-cadherin antibodies.
  • CD31 + isolated cells was identified between passage 0 to passage 1 using antibodies against endothelial markers: CD31 and VEGFRl.
  • CD31+/VEGFR1+ PSC-derived endothelial cells comprised the majority of cell population as opposed to the non- endothelial cells (ECs) CD31-/VEGFR1- population ( Figure 26A).
  • FIGs. 27 A-E are microscopy images ( Figures 27A-D) and a dot plot ( Figure 27E) demonstrating that cultured CD105+/CD31+ PSC-derived cells exhibit stable endothelial characteristics in vitro.
  • Isolated CD105+/CD31+ cultured in M- 199 supplemented with 20% FBS, 50 ⁇ g/ml ECGS and 20 U/ml heparin present features of vasculogenic endothelial in vitro including: tube formation on Matrigel (Figure 27A), stable expression of specific endothelial cell markers UEA-I ( Figure 27B) or CD31 ( Figure 27C) and could be expanded up to passage 5 ( Figure 27D).
  • CD105 + endothelial cells were isolated from IxIO 8 cells of dissociated iPSC-derived EBs and expanded as follows: 3.5xlO 6 after second isolation step from pericytes- endothelial cells mixed culture, 7xlO 6 at passage 2, 1.5xlO 7 at passage 3, 5xlO 7 at passage 4, and IxIO 8 at passage 5.
  • Flow cytometry analysis shows that cultivated CD105 + endothelial cells maintained CD31 expression (Figure 27E).
  • FIG. 28 is a dot plot depicting flow cytometry analysis of CD31 of EBs cells generated from human foreskin-derived iPSCs demonstrating derivation of endothelial cells from iPSC.
  • Human foreskin fibroblasts were used for generation of iPSC.
  • Dissociated EBs from iPSC (14 days old) contain CD31 + endothelial population.
  • FIGs. 29 A-F are microscopy images depicting immunofluorescence of CD105+ cells-derived pericytes. Cultivated CD105+/CD31- PSC-derived pericytes exhibit pericytic markers in vitro. Immuno-labeling of cultured pericytes revealed co- expression of Calponin (red, Figures 29A and 29D) and NG2 (green, Figures 29B and 29E). Figures 29C and 29F display merged images of calponin, NG2 with nuclear staining with DAPI (blue). Original magnifications: x200 in Figures 29 A-C; and x630 in Figures 29D-F.
  • FIGs. 30A-C are dot plots ( Figure 30A-B) and a microscopy image ( Figure 30C) depicting representative phenotypes of cultured pericytes from passage 1 to passage 9 or during senescence.
  • Figure 3OA flow cytometry of co-cultured CD105+ cells at passage 0 (PO) using CD31 and ⁇ SMA antibodies
  • Figure 3OB flow cytometry of pure CD105+ pericytes at passage 4 (P4) using CD31 and CD105 antibodies
  • Figure 3OC - immunofluorescence of CD105+/CD31- cells from passage P6) using NG2 (green) and Claponin (red). Nuclei are stained with DAPI (blue).
  • FIGs. 31A-B are images depicting tube formation and sprouting on Matrigel.
  • Pericytes form tubular network when seeded on Matrigel with sprouting clusters (arrows) within 12 hours ( Figure 31A). Morphology of pericytes at passage 4 ( Figure 31B). The pericyte cells exhibit high expansion capability, e.g., from 5xlO 5 in passage 0 (PO) to 4xlO 7 in P4 (passage 4).
  • FIGs. 32A-C are images depicting implants in immuno-deficient NOD/SCID mice.
  • PSC-derived pericytes from passage 6 were mixed in Matrigel or empty Matrigel were implanted subcutaneously into immune-deficient mice.
  • implants containing H9.2-derived pericytes are vascularized ( Figure 32A, left) in comparison to empty control Matrigel implant ( Figure 32A, right).
  • Empty implants do not contain blood vessels ( Figures 32B and 32C) as seen by Hematoxylin and Eosin staining.
  • FIGs. 33A-D are microscopy images depicting pericyte-Matrigel implants in immunodefficient NOD/SCID mice.
  • PSC-derived pericytes from passage 6 were mixed in Matrigel and implanted subcutaneously into immune-deficient mice.
  • implants containing H9.2-derived pericytes contain murine blood vessels as seen by Hematoxylin and Eosin staining ( Figures 33A and 33B).
  • all PSC-derived pericytes further differentiate into smooth muscle cells, which express ⁇ -SMA (red) after implantation ( Figures 33C and 33D).
  • Nuclear staining by DAPI blue).
  • Original magnifications xlOO in Figures 33A and 33C; x200 in Figures 33B and 33D.
  • FIGs. 34A-B are microscopy images depicting immunofluorescence analyses of newly formed vasculaturb within Matrigel implants. Differentiated ⁇ -SMA (red) positive PSC-derived pericytes are seen incorporated to newly formed murine blood vessels within Matrigel implant ( Figures 34A and 34B). Nuclear staining by DAPI appears in blue ( Figure 34A). Original magnifications: x630.
  • FIG. 35 is an image depicting tube like formation by pericytes and endothelial cells on Matrigel with pericyte sprouting. Cultured PSC-derived pericyets and endothelial cells were re-mixed post their separate expansion. Note the assembly of the cells to form tube like structures on Matrigel in vitro.
  • FIGs. 36A-C are microscopy images depicting Hematoxylin and Eosin staining of Matrigel implants of endothelial cells and pericytes.
  • Matrigel was mixed with H9.2- derived pericytes and endothelial cells and implanted subcutaneously into immune- deficient mice. Implants were harvested after one week.
  • Hematoxylin and Eosin staining reveals the presence of erythrocytes containing blood vessels (indicated by arrows) surrounded by Matrigel islands (M) and clusters of PSC-derived pericytes (Figures 36A-36C).
  • Original magnifications xlOO in Figure 36A; x200 in Figures 36B and 36C.
  • FIG. 37 is a graph depicting expansion of CD31+ cells in dissociated EBs using various dissociation methods.
  • Single cell suspensions were achieved by incubation of EBs for 20 minutes at 37°C oh shaker with either (1) 0.5% Trypsin/EDTA (Sigma), (2) non-enzymatic solution (Sigma) or (3) 1 mg/ml collagenase B in PBS and 150 U/ml DNAse I (Roche), followed by addition of Trypsin-EDTA (0.05%) for another 5 minutes.
  • Trypsin-EDTA 0.5% Trypsin/EDTA
  • Sigma non-enzymatic solution
  • Roche U/ml DNAse I
  • Collagenase B+ DNAse I method revealed a higher dissociation efficiency than the Trypsin method or the non- enzymatic solution-based method.
  • viability of the cells dissociated using the Collagenase B+ DNAse I method was 95% as compared to only 50-60% using the other methods (Trypsin or the non-enzymatic solution).
  • FIG. 38 is a microscopy image of myotubes in culture generated by differentiation of iPSC C3-derived pericytes.
  • FIGs. 39A-F are dot plots depicting flow cytometry analyses showing the expression of CD73 and CD31 in cells derived from developing EBs.
  • EBs were spontaneously formed (differentiated) from induced pluripotent stem cells (Figures 39A-F) and FACS analysis was performed at the indicated days of EBs differentiation: Day 1 ( Figure 39A), day 4 ( Figure 39B), day 10 (Figure 39C), day 14 ( Figure 39D), day 19 ( Figure 39E), and day 26 ( Figure 39F).
  • the Y axis in each panel represents CD73 expression in the dissociated EBs single cells
  • the X axis in each panel represents CD31 expression in the dissociated EBs single cells.
  • H9.2 hESC-derived developing EBs were analyzed similarly and exhibit similar pattern of marker expressions (data not shown).
  • the present invention in some embodiments thereof, relates to isolated pericyte progenitor cells and methods of generating and using same, and more particularly, but not exclusively, to methods of co-derivation two distinguished vasculogenic populations, pericytes and endothelial cells, from pluripotent stem cells and embryoid bodies.
  • the present inventors have isolated vascular progenitor cells from differentiating embryoid bodies generated from either human embryonic stem cells or induced pluripotent stem cells. Isolated CD105+ cells were subject to co-derivation into pericyte progenitor cells having the CD105+/CD73+/CD31- expression signature, and endothelial progenitor cells having the CD105+/CD73+/CD31+ expression signature ( Figures 1-3, 21-24, 26, 27, 39; Examples 1, 2, 6 and 7 of the Examples section which follows).
  • the isolated pericyte progenitor cells exhibit a unique expression pattern characterized by the CD105+/CD73+CD31-/ ⁇ SMA-/CD133- /FIk-I- signature.
  • the pericyte progenitor cells were shown capable of assembly into a human vascular network ( Figures 15-18, 31, 32, 35 and 36, Examples 4 of the Examples section which follows), as well as to differentiate to multiple cell types of the mesenchymal lineage including differentiation into osteoblasts both in vitro and in vivo ( Figures 19A-F, Example 5 of the Examples section which follows), chondrocytes (Example 5 of the Examples section which follows), adipocytes ( Figures 20A-B, Example 5 of the Examples section which follows), myoblasts ( Figure 38, Example 5 of the Examples section which follows) and to smooth muscle cells ( Figures 33C-D, 34A- B, Example 5 of the Examples section which follows).
  • a method of isolating a pericyte progenitor cell from embryoid bodies is effected by: (a) isolating CD105+, CD73+ and/or CD105+/CD73+ cells from the embryoid bodies, to thereby obtain CD105+, CD73+ and/or CD105+/CD73+ cells, and; (b) culturing the CD105+, CD73+ and/or CD105+/CD73+ cells, thereby isolating the pericyte progenitor cell from the embryoid bodies.
  • pericyte progenitor cell refers to an immature pericyte cell (i.e., a more prir ⁇ itive cell in the hierarchy of pericyte differentiation) which is capable of further differentiation into cell type(s) of a mesenchymal lineage.
  • Examples of cells originating from a mesenchymal lineage include, but are not limited to chondrocytes, osteoblasts, adipocytes and myoblasts.
  • Pericyte cells also known as Rouget cell, adventitial cell or mural cell, are perivascular cells present in small blood vessels. Pericytes are characterized by the expression of NG2 [chondroitin sulfate proteoglycan 4, official gene symbol: CSPG4, also known as MCSP; MCSPG; MSK16; HMW-MAA; MEL-CSPG] and PDGFR- ⁇ [platelet-derived growth factor receptor, beta polypeptide, official gene symbol: PDGFRB, also known as JTK12; PDGFR; CD140B; PDGFRl] (Ugur Ozerdem and William B. Stallcup, Angiogenesis. 2003; 6: 241-249).
  • NG2 chondroitin sulfate proteoglycan 4, official gene symbol: CSPG4, also known as MCSP; MCSPG; MSK16; HMW-MAA; MEL-CSPG
  • PDGFR- ⁇ platelet-derived growth factor receptor, beta polypeptide, official gene symbol: PDGFRB, also known
  • ⁇ SMA alpha smooth muscle actin
  • ACTA2 also known as AAT6
  • ACTSA a late marker for differentiated pericytes in rodents and therefore may be poorly expressed in developing angiogenic microvasculature (Ugur Ozerdem and William B. Stallcup, Angiogenesis. 2003; 6: 241- 249).
  • embryonic bodies refers to three dimensional multicellular aggregates of differentiated and undifferentiated cells derivatives of three embryonic germ layers.
  • Embryoid bodies are formed upon the removal of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) from feeder layers or feeder cells-free culture systems.
  • ESCs and/or iPSCs removal can be effected using type IV Collagenase treatment for a limited time.
  • the cells are transferred to tissue culture plates containing a culture medium supplemented with serum and amino acids.
  • EBs are further monitored for their differentiation state.
  • Cell differentiation can be determined upon examination of cell or tissue-specific markers which are known to be indicative of differentiation.
  • EB-derived- differentiated cells may express the neurofilament 68 KD which is a characteristic marker of the ectoderm cell lineage.
  • the differentiation level of the EB cells can be monitored by following the loss of expression of Oct-4, and the increased expression level of other markers such as ⁇ - fetoprotein, NF-68 kDa, ⁇ -cardiac and albumin.
  • Methods useful for monitoring the expression level of specific genes are well known in the art and include RT-PCR, semiquantitative RT-PCR, Northern blot, RNA in situ hybridization, Western blot analysis and immunohistochemistry.
  • Embryoid bodies can be generated from various primates and mammals such as human, monkeys and rodents (e.g., mouse, rat).
  • the embryoid bodies are obtained from human embryoid bodies.
  • the embryoid bodies are obtained by spontaneous differentiation of pluripotent stem cells.
  • embryonic stem cells refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (Le., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state.
  • embryonic stem cells may read on cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post- implantation/pre-gastrulation stage blastocyst (see WO2006/040763) and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.
  • embryonic tissue formed after gestation e.g., blastocyst
  • EBCs extended blastocyst cells
  • EG embryonic germ
  • the embryonic stem cells of some embodiments of the invention can be obtained using well-known cell-culture methods.
  • human embryonic stem cells can be isolated from human blastocysts.
  • Human blastocysts are typically obtained from human in vivo preimplantation embryos or from in vitro fertilized (IVF) embryos.
  • IVF in vitro fertilized
  • a single cell human embryo can be expanded to the blastocyst stage.
  • the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting.
  • ICM inner cell mass
  • the ICM is then plated in a tissue culture flask containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re-plated on a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and reflated. Resulting ES cells are then routinely split every 4-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol.
  • ES cells can also be used with this aspect of the present invention.
  • Human ES cells can be purchased from the NIH human embryonic stem cells registry (www.escr.nih.gov).
  • Non-limiting examples of commercially available embryonic stem cell lines are BGOl, BG02, BG03, BG04, CY12, CY30, CY92, CYlO, TE03 and TE32.
  • ES cells can be obtained from other species as well, including mouse (Mills and Bradley, 2001), golden hamster [Doetschman et al., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, Dev Biol. 163: 288-92] rabbit [Giles et al. 1993, MoI Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, MoI Reprod Dev. 1993, 36: 424-33], several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev.
  • EBCs Extended blastocyst cells
  • EBCs can be obtained from a blastocyst of at least nine days post fertilization at a stage prior to gastrulation.
  • the zona pellucida Prior to culturing the blastocyst, the zona pellucida is digested [for example by Tyrode's acidic solution (Sigma Aldrich, St Louis, MO, USA)] so as to expose the inner cell mass.
  • the blastocysts are then cultured as whole embryos for at least nine and no more than fourteen days post fertilization (Le., prior to the gastrulation event) in vitro using standard embryonic stem cell culturing methods.
  • EG cells are prepared from the primordial germ cells obtained from fetuses of about 8-11 weeks of gestation (in the case of a human fetus) using laboratory techniques known to anyone skilled in the arts.
  • the genital ridges are dissociated and cut into small chunks which are thereafter disaggregated into cells by mechanical dissociation.
  • the EG cells are then grown in tissue culture flasks with the appropriate medium.
  • the cells are cultured with daily replacement of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.
  • Induced pluripotent stem cells are cells obtained by de-differentiation of adult somatic cells which are endowed with pluripotency (i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm).
  • pluripotency i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm.
  • such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin) and undergo de-differentiation by genetic manipulation which re-program the cell to acquire embryonic stem cells characteristics.
  • the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell.
  • iPS cells can be generated by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, gastric epithelial cells with transcription factors such as Oct-3/4, Sox2, c- Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007, l(l):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14.
  • embryonic-like stem cells can be generated by nuclear transfer to oocytes, fusion with embryonic stem cells or nuclear transfer into zygotes if the recipient cells are arrested in mitosis.
  • isolating the pericyte progenitor cell is effected using pluripotent stem cells, which are induced to form embryoid bodies prior to isolation of the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the embryoid bodies or the pluripotent stem cells are derived from an individual having a normal karyotype according to the species to which the individual belong.
  • a normal karyotype is of 22XY or 22XX chromosomes.
  • the embryoid bodies or the pluripotent stem cells are derived from a healthy individual.
  • embryoid bodies or the pluripotent stem cells are derived from an individual having a disease or a pathology, such as a pathology related to an impaired, dysfunction, absence or destroyed vascular tissue.
  • a disease or a pathology such as a pathology related to an impaired, dysfunction, absence or destroyed vascular tissue.
  • Non-limiting examples include individuals having ischemia, diabetes, diabetic microangiopathy, peripheral arterial disease, cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis, vascular injury, vascular repair due to removal of cancerous tissue and the surrounding vasculature, tissue fibrosis, cancer, Alzheimer's disease, chronic lung disease, trauma, injury, cancer, diabetes, blood coagulation related- disorders (e.g., over coagulation).
  • the embryoid bodies are cultured on defined, xeno-free, feeder-free culturing systems.
  • feeder-free systems e.g., using low attachment culture dishes
  • a culture medium which is serum-free, and/or xeno-free (i.e., devoid of contamination by another species, for example, devoid of animal contamination of human cells) provides a more defined environment for the EBs, which can be controlled, such as to be free of xeno- contaminant and cellular contaminants.
  • Isolating the CD105+ (cells which express CD105), the CD73+ (cells which express CD73), and/or the CD105+/CD73+ (cells which express both CD105 and CD73 markers) from the embryoid bodies can be performed at any EBs differentiation stage, as long as the EBs include CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the fraction of CD105+ or CD73+ cells significantly increases between day 4 and 7, and further between day 7 to 10 and 10 to 14 of EBs differentiation.
  • the fraction of CD105+/CD31- cells increases between day 14 and 26 of EBs differentiation.
  • isolating CD105+, CD73+ and/or CD105+/CD73+ cells is performed between about day 4 to about day 26 of EBs differentiation, e.g., between about day 7 to about day 26 of EBs differentiation, e.g., between about day 10 to about day 26 of EBs differentiation, e.g., between about day 14 to about day 26 of differentiation of the embryoid bodies.
  • the first day of EBs differentiation is considered about 24 hours after the pluripotent stem cells were allowed to differentiate in vitro by removing the pluripotent stem cells from their undifferentiation culture conditions, such as by removing them from feeder layers or from their feeder-free culture systems (e.g., matrix such as an extracellular matrix).
  • their undifferentiation culture conditions such as by removing them from feeder layers or from their feeder-free culture systems (e.g., matrix such as an extracellular matrix).
  • Isolating the CD105+, CD73+ and/or CD105+/CD73+ cells from the embryoid bodies can be performed by any immunological based method which results in the physical isolation of cells having a specific cell surface marker using an antibody or an antibody fragment which specifically recognizes the marker. Examples include, but are not limited to isolation by fluorescence-activated cell sorting using the specific antibodies, magnetic beads coated by the specific antibodies, and columns coated by the specific antibodies.
  • CD 105 also known as endoglin (gene symbol ENG) is a homodimeric transmembrane protein, a major glycoprotein of the vascular endothelium. Endoglin is a component of the transforming growth factor beta receptor complex and it binds TGFBl and TGFB3 with high affinity. There are two known variants of endoglin: isoform 1 (GenBank Accession No. NP_001108225.1; SEQ ID NO:17) and isoform 2 (GenBank Accession No. NP_000109.1; SEQ ID NO:18).
  • isolating the CD105+ and/or the CD105+/CD73+ cells is effected using an anti CD105 antibody.
  • Suitable CD105 antibodies which can be used to isolate the CD105+ or CD105+/CD73+ cells from the EBs include R-Phycoerythrin (PE)-conjugated anti- CD 105 (eBioscience), Fluorescein isothiocyanate (FITC) -conjugated anti-CD 105 (ABCAM), APC-conjugated anti-CD105 (eBioscience).
  • PE Physically-Phycoerythrin
  • FITC Fluorescein isothiocyanate
  • ABCAM Fluorescein isothiocyanate
  • APC-conjugated anti-CD105 eBioscience.
  • Ecto-5-prime-nucleotidase also known as NT; eN; NT5; NTE; eNT; E5NT; NT5E (EC 3.1.3.5; GenBank Accession No. NP_002517.1; SEQ ID NO:19), catalyzes the conversion at neutral pH of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP.
  • the enzyme consists of a dimer of 2 identical 70-kD subunits bound by a glycosyl phosphatidyl inositol linkage to the external face of the plasma membrane.
  • isolating the CD73+ and/or CD105+/CD73+ cells is effected using a CD73 antibody.
  • Suitable CD73 antibodies which can be used to isolate the CD73+ or CD73+/CD105+ cells from the EBs include PE-conjugated anti-CD73 (BD Pharmingen), FITC-conjugated anti-CD73 (eBioscience), APC conjugated anti-CD73
  • the cells are labeled with a fluorescent antibody (e.g., PE-conjugated anti CD105 antibody, or PE-conjugated anti CD73 antibody) and then inserted into a cell sorter (e.g., FACS Aria sorter).
  • a fluorescent antibody e.g., PE-conjugated anti CD105 antibody, or PE-conjugated anti CD73 antibody
  • a cell sorter e.g., FACS Aria sorter
  • the cells are labeled with a magnetic bead conjugated antibody anti CD105 antibody (Miltenyi Biotec) or anti CD73 antibody; alternatively, the cells can be labeled with a non-conjugated antibody and followed by incubation with a match isotype bead conjugated secondary antibody (anti mouse IgGl bead conjugated). Isolation is performed using magnetic cell separation column such as MAX (Miltenyi Biotec).
  • the EBs are dissociated to separate the cell clumps and cell aggregates into single cells.
  • the dissociation is performed under conditions which enable separation of cell aggregates/clumps while preserving the viability of the separated cells of the dissociated EBs.
  • the dissociation of EBs is performed by treatment with Collagenase and DNAse I.
  • the Collagenase can be Collagenase B (e.g., available from Roche, catalogue number 11 088 807 001) used in a concentration in the range of about 0.1-5 mg/ml, e.g., about 0.5-3 mg/ml, e.g., about 0.8-2 mg/ml, e.g., about 0.8-1.5 mg/ml, e.g., about 1 mg/ml Collagenase B.
  • the DNAse I (e.g., available from Roche, catalogue number 2139) can be used in a concentration of about 10-500 U/ml, e.g., about 50-350 U/ml, e.g., about 100-200 U/ml, e.g., about 150 U/ml DNAse I.
  • Incubation with the Collagenase and DNAse I solution can be performed while shaking the vessel containing the EBs, for an incubation time which may vary between about 5-30 minutes, e.g., between about 10-25 minutes, e.g., between about 15-20 minutes.
  • the dissociation can be performed at about 37°C while shaking. Trypsin may be added for a limited time (e.g., about 5 minutes) and concentration (e.g., a solution of about 0.05%).
  • concentration e.g., a solution of about 0.05%).
  • measures are taken to avoid cell damage, and the dissociation conditions can be adjusted according to the source or origin of the EBs.
  • the dissociated EBs can be further passed several times through a 20-Gauge needle and filtered through a cell-strainer (e.g., PBS/0.5% fetal bovine serum (FBS) pre-washed 0.45 cell-strainers (BD Biosciences).
  • a cell-strainer e.g., PBS/0.5% fetal bovine serum (FBS) pre-washed 0.45 cell-strainers (BD Biosciences).
  • the isolation step with the anti-CD105 antibody results in a population of cells which comprises at least about 85% of CD105+ or CD105+/CD73+ cells, e.g., at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of cells which are CD105+ or CD105+/CD73+.
  • the isolation step with the ariti-CD73 antibody (or antibodies) results in a population of cells which comprises at least about 85% of CD73+ or CD105+/CD73+ cells, e.g., at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of cells which are CD73+ or CD1Q5+/CD73+.
  • the present inventors demonstrated the isolation of pericyte progenitor cells by enriching the CD105+/CD31- cells (i.e., cells which express CD105 and which do not express CD31) in the isolated population of cells.
  • the method further comprising enriching the cells for CD105+/CD31-, CD73+/CD31- and/or CD105+/CD73+/CD31- cells.
  • the phrase "enriching ... cells” refers to increasing the percentage of cells characterized by a specific marker expression signature in a heterogenous population of cells.
  • the heterogenous population of cells isolated from EBs includes CD105+, CD73+ and/or CD105+/CD73+ cells which are either CD31+ or CD31-, the enrichment step results in a majority of cells which are
  • the enrichment step results in a population of cells which comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% of cells which are CD105+/CD31-, CD73+/CD31- and/or CD105+/CD73+/CD31-.
  • enriching the cells is effected prior to culturing the isolated CD105+, CD73+ and/or CD105+/CD73+ cells from the EBs.
  • enriching is performed by depleting CD31+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the phrase "depleting ... cells” refers to decreasing the percentage of cells characterized by a specific marker expression signature in a heterogenous population of cells and/or removing cells characterized by the specific marker expression signature from a heterogenous population of cells, such that the population of cells is devoid of the depleted cells.
  • a heterogenous population of cells comprises both CD31+ and
  • CD31- cells then depleting the CD31+ cells results in significantly decreasing the percentage of CD31+ from the heterogenous population and optionally, completely removing the CD31+ cells from the heterogenous population in order to obtain a population which is characterized by fhe CD31- expression signature.
  • the depletion step results in a population of cells which comprises no more than 20%, e.g., no more than 19%, 18%,
  • Depleting CD31+ cells can be performed by fluorescence-activated cell sorting or using magnetic beads as described above, except that the cells which are CD31+ are removed from the cell population and the cells which are CD31- are retrieved (isolated).
  • the method further comprising passaging the CD105+, CD73+ and/or CD105+/CD73+ cells for at least 1-2 passages to thereby expand a population of pericyte progenitor cells.
  • Culturing of the isolated CD105+, CD73+ and/or CD105+/CD73+ cells can be performed in a two-dimensional culture system by seeding the cells on a surface of a culture dish (e.g., a plastic dish, a flask).
  • a culture dish e.g., a plastic dish, a flask
  • the CD105+, CD73+ and/or CD105+/CD73+ cells can be cultured on coated plates/flasks to increase adhesiveness of the cells.
  • the cells can be cultured without further coating of tissue culture dishes (plates/flasks).
  • the culture dish can be coated with an extracellular matrix, preferably a synthetic or xeno-free extracellular matrix.
  • Non-limiting examples of matrices which can be used include foreskin matrix, laminin matrix, fibronectin matrix, proteoglycan matrix, entactin matrix, heparan sulfate matrix, collagen matrix and the like, alone or in various combinations thereof.
  • the matrix is xeno-free.
  • xeno is a prefix based on the Greek word “Xenos", i.e., a stranger.
  • xeno-free refers to being devoid of any components which are derived from a xenos (i.e., not the same, a foreigner) species. Such components can be contaminants such as pathogens associated with (e.g., infecting) the xeno species, cellular components of the xeno species or a-cellular components (e.g., fluid) of the xeno species.
  • the matrix is preferably derived from a human source or synthesized using recombinant techniques such as described hereinabove.
  • Such matrices include, for example, human-derived fibronectin, recombinant fibronectin, human-derived laminin, foreskin fibroblast matrix or a synthetic fibronectin matrix.
  • Human derived fibronectin can be from plasma fibronectin or cellular fibronectin, both of which can be obtained from Sigma, St. Louis, MO, USA.
  • Human derived laminin and foreskin fibroblast matrix can be obtained from Sigma, St. Louis, MO, USA.
  • a synthetic fibronectin matrix can be obtained from
  • the matrix is a human fibronectin matrix.
  • CD105+/CD73+ cells can be an endothelial cell growth medium, such as an M-199 based culture medium (available from Biological Industries, Israel).
  • the medium can be supplemented with serum (e.g., human serum, bovine serum, or serum replacement) and additional additives such as an endothelial cell growth supplement (ECGS) (available from Zotal Biological & Instrumentation, Israel).
  • serum e.g., human serum, bovine serum, or serum replacement
  • additional additives such as an endothelial cell growth supplement (ECGS) (available from Zotal Biological & Instrumentation, Israel).
  • an endothelial cell medium which can be used include the M-199 medium containing 20% defined-fetal bovine serum (FBS) (HyClone, Utah, USA), 1% Pen-Strep, 1% 1- glutamine, 1 mM HEPES (Biological Industries), 20 U/ml heparin (Sigma-Aldrich) and 50 ⁇ g/ml endothelial cell growth supplement (ECGS) (Zotal).
  • FBS defined-fetal bovine serum
  • Pen-Strep 1% 1- glutamine
  • 1 mM HEPES Biological Industries
  • 20 U/ml heparin Sigma-Aldrich
  • ECGS endothelial cell growth supplement
  • the medium is xeno-free and comprises either human serum or a xeno-free serum replacement.
  • a xeno-free serum replacement can include a combination of insulin, transferrin and selenium.
  • a xeno-free serum replacement can include human or recombinantly produced albumin, transferrin and insulin.
  • Passaging can be performed by dissociating cells from the wall of the culture vessel using e.g., type IV collagenase (at a concentration of 0.1 % for 20-60 minutes) followed by trypsinization (using 0.25 % trypsin for 2-5 minutes), counting the single cells and splitting the cells to 2-3, (i.e., a splitting ratio of 1:2 or 1:3) in order to preserve the same cell density of their initial seeding (e.g., about 5 x 10 5 - 1 x 10 6 cells per 15 ml in T75 flasks).
  • the cell culture is subjected to culture passaging every 2-8 days, e.g., culture passaging occurs every 3-8 days, e.g., every 4 days.
  • Culturing can be performed for several passages, e.g., from 1-9 passages, e.g., 1- 2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 7-9 passages, or until senescence.
  • 1-9 passages e.g., 1- 2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 7-9 passages, or until senescence.
  • the term "senescence” refers to the stage in which the cells lose their ability to divide. According to some embodiments of the invention, even in the absence of an active enrichment step, following about 1-2 passages at least 96%, 97%, 98%, 99% or 100% of the cells having a signature of CD105+/CD73+/CD31-.
  • pericyte progenitor cells can be isolated from EBs in either way, i.e., with or without the active enrichment of CD31- cells.
  • the CD105+/CD73+/CD31- cells can be further cultured in order to expand the pericyte progenitor ceil population.
  • the cells isolated from EBs according to the method of some embodiments of the invention exhibit pericyte characteristics in term of expression of NG2 and PDGFR- ⁇ .
  • the pericyte progenitor cells while in culture (e.g., in a two dimensional culture dish), are substantially free of contact inhibition.
  • the pericyte progenitor cells adopt a hill and valley morphology.
  • an isolated population of cells which comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100% of pericyte progenitor cells having the CD105+/CD31-/ ⁇ SMA-, a CD73+/CD31-/ ⁇ SMA- or a CD105+/CD73+/CD31-/ ⁇ SMA- signature.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD31- and/or CD73+/CD31-.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD133- and/or CD73+/CD133-.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/NG2+ and/or CD73+/NG2+.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD31-/CD133- and/or CD73+/CD31-/CD133- cells.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of .
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD146+ and/or CD73+/CD146+.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD90+ and/or CD73+/CD90+.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD31-/ ⁇ SMA-/CD146+/CD90+ and/or CD73+/CD31- / ⁇ SMA-/CD146+/CD90+.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD31-/CD133- and/or CD73+/CD31-/CD133- cells.
  • At least about 90% e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells isolated by the method of some embodiments of the invention having an expression marker signature of CD105+/CD31-/CD133-/NG2+ and/or CD73+/CD31-/CD133- /NG2+ cells.
  • the population of cells generated by the method of some embodiments of the invention maintains the expression signature of the isolated cells before culturing and/or before passaging.
  • the population of cells comprises at least about 85%, e.g., at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more of cells having an expression marker signature of CD105+/CD31-/ ⁇ SMA- and/or CD73+/CD31-/ ⁇ SMA-.
  • the population of cells comprises at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more of cells having a CD105+/CD73+/CD31-/ ⁇ SMA-/CD133-/Flk- /NG2+/CD146+/CD90+/Tie-l+/Tie-2+ signature.
  • an isolated pericyte progenitor cell there is provided an isolated pericyte progenitor cell.
  • the phrase "isolated” refers to being at least partially separated from the natural environment e.g., the embryoid bodies or from non-pericyte progenitor cells which are present in the EBs. It should be noted that the term “cell” may also read on a plurality of cells (e.g., a cell line), wherein all of the cells have at least about the same expression marker signature as that of the single cell.
  • the isolated pericyte progenitor cell is separated from other cells of the EBs, such as cells having a different expression marker signature than that of the isolated pericyte progenitor cell.
  • the isolated pericyte progenitor cell is separated from cells having an expression marker signature of CD31- /CD105- or CD105+/CE>31-/Flk-l+.
  • the isolated pericyte progenitor cell when the isolated pericyte progenitor cell is comprised in a plurality of cells, the plurality of cells is devoid of CD31-/CD105- cells and/or being devoid of CD105+/CD31-/Flk-l+ cells.
  • the isolated pericyte progenitor cell having a CDlO5+/CD73+/CD31-/ ⁇ SMA- signature.
  • the isolated pericyte progenitor cell having a CD105+/CD31-/ ⁇ SMA-/CD133-, a CD73+/CD31-/ ⁇ SMA- /CD133- or a CD105+/CD73+CD31-/OSMA-/CD133- signature.
  • the isolated pericyte progenitor cell having a CDIOSH-ZCDSl-ZaSMA-ZCDlSS-ZFIk-I-, a CD73+ZCD31- Z ⁇ SMA-ZCDm-ZFlk-l- or a CD105+ZCD73+CD31-Z ⁇ SMA-ZCD133-ZFlk-l- signature.
  • the pericyte progenitor cell having a CD105+ZCD73+ZCD31-Z ⁇ SMA-ZCD133- signature.
  • the pericyte progenitor cell having a CD105+ZCD73+ZCD31-Z ⁇ SMA-ZFlkl- or CD105+ZCD73+ZCD31-Z ⁇ SMA- ZCD133-ZFlkl- signature.
  • the pericyte progenitor has an expression marker signature of NG2+Z ⁇ SMA-.
  • the pericyte progenitor cell having a CD105+ZCD73+ZCD31-Z ⁇ SMA-ZNG2+, CD105+ZCD73+ZCD31-Z ⁇ SMA- ZCD133-ZNG2+ or CD105+ZCD73+ZCD31-Z ⁇ SMA-ZCD133-ZFlkl-ZNG2+ signature.
  • the pericyte progenitor cell is CD146+.
  • Such a pericyte cell can have an expression marker signature of CD105+/CD73+/CD31-/o ⁇ SMA-/Flkl-/CDl46+, CD105+/CD73+/CD31-/ ⁇ SMA-/Flkl-
  • CD146+ CD105+/CD73+/CD31-/ ⁇ SMA-/NG2+/CD146+, CD105+/CD73+/CD31- / ⁇ SMA-/CD133-/NG2+/CD146+, or CD105+/CD73+/CD31-/ ⁇ SMA-/CD133-/Flkl- /NG2+/CD146+ signature.
  • the pericyte progenitor cell is
  • Such a pericyte cell can have an expression marker signature of CD105+/CD73+/CD31-/ ⁇ SMA-/Flkl -/CD90+, CD105+/CD73+/CD31-/ ⁇ SMA-
  • the pericyte progenitor cell is
  • Such a pericyte cell can have an expression marker signature of CD105+/CD73+CD31-/ ⁇ SMA-/CD133-/Flk-l-/Tie-l+/Tie-2+, CD105+/CD73+/CD31- / ⁇ SMA-/CD133-/Tie-l+/Tie-2+, CD105+/CD73+/CD31-/ ⁇ SMA-/Flkl-/Tie-l+/Tie-2+, CD105+/CD73+/CD31-/ ⁇ SMA-/CD133-/Flkl-/Tie-l+/Tie-2+, CD105+/CD73+/CD31- / ⁇ SMA-/NG2+/Tie-l+/Tie-2+, CD105+/CD73+/CD3 l-/ ⁇ SMA-/CD133-/NG2+/Tie- l+/Tie-2+, CD105+/CD73+/CD3 l
  • CD105+/CD73+/CD31-/ ⁇ SMA-/Flkl-/CD90+/Tie-l+/Tie-2+ CD105+/CD73+/CD31- / ⁇ SMA-/CD133-/Flkl-/CD90+/Tie-l+/Tie-2+, CD105+/CD73+/CD31-/ ⁇ SMA-
  • the pericyte progenitor cell is capable of differentiation into at least two cell lineages of the cell lineages selected from the group consisting of osteoblasts, chondrocytes, myobloasts, smooth muscle cells and apipocytes.
  • the pericyte progenitor cells were capable of differentiation into osteoblasts ( Figures 19A-B and Example 5 of the Examples section which follows), adipocytes ( Figures 20A-B and Example 5 of the Examples section which follows), chondrocytes (Example 5 of the Examples section which follows) myoblasts ( Figures 33A-D, 38 and Example 5 of the Examples section which follows) and smooth muscle cells ( Figures 33C-D, 34A-B, Example 5 of the Examples section which follows).
  • the pericyte progenitor cell is capable of differentiation into at least three, four or five cell lineages of the cell lineages selected from the group consisting of osteoblasts, chondrocytes, myobloasts, smooth muscle cells and apipocytes.
  • the ability of the pericyte progenitor cell to differentiate into at least two cell lineages of the cell lineages selected from the group consisting of osteoblasts, chondrocytes, myobloasts, smooth muscle cells and apipocytes is maintained at any passage in culture from passage 1 to senescence.
  • the pericyte progenitor is not genetically modified to express an exogenous gene such as a reporter gene (e.g., green fluorescence protein, GFP).
  • a reporter gene e.g., green fluorescence protein, GFP
  • a cell culture comprising a culture medium and the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention.
  • the medium can be any liquid medium suitable for culturing the pericyte progenitor cells and maintaining them in an undifferentiated state (i.e., capable of multipotent differentiation).
  • Non-limiting examples of such a medium include, but are not limited to an M-199 based culture medium (available from Biological Industries, Israel, Catalogue No. 01-085-1A).
  • the medium can be supplemented with serum (e.g., human serum, bovine serum, or serum replacement) and additional additives such as an endothelial cell growth supplement (ECGS) (available from Zotal Biological & Instrumentation, Israel).
  • serum e.g., human serum, bovine serum, or serum replacement
  • additional additives such as an endothelial cell growth supplement (ECGS) (available from Zotal Biological & Instrumentation, Israel).
  • ECGS endothelial cell growth supplement
  • a method of generating osteoblast cells (osteogenic differentiation).
  • the method is effected by culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in an osteogenic medium such as a culture medium which comprises ⁇ -glycerol- phosphate, Dexamethasone and ascorbic acid, thereby generating the osteoblast cells.
  • the pericyte progenitor cells are grown in the presence of a culture medium containing 10 mM ⁇ -glycerol-phosphate and 0.1 ⁇ M Dexamethasone in GMEM BHK-21 medium (which comprises ascorbic acid) (Gibco) containing 10 % FBS for 4 weeks, with media changes twice a week.
  • GMEM BHK-21 medium which comprises ascorbic acid (Gibco) containing 10 % FBS for 4 weeks, with media changes twice a week.
  • Cell cultures can be assayed for mineral content by Alizarin red staining.
  • the pericyte progenitor cells are grown in the presence of a culture medium containing alpha-MEM (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 15 % FBS (selected lots, Hyclone), 50 ⁇ g/ml ascorbic acid (Sigma, St Louis, MO, USA), 10 ⁇ 7 M dexamethasone (Sigma, St Louis, MO, USA) and 10 mM beta-glycerophosphate (inorganic phosphate), and let become over-confluent for period of at least 10 days before mineralization appears.
  • alpha-MEM Biological Industries, Kibbutz Beit Haemek, Israel
  • FBS selected lots, Hyclone
  • 50 ⁇ g/ml ascorbic acid Sigma, St Louis, MO, USA
  • 10 ⁇ 7 M dexamethasone Sigma, St Louis, MO, USA
  • 10 mM beta-glycerophosphate inorganic phosphate
  • the osteoblast cells are characterized by mineral deposits and massive calcium content.
  • a method of generating adipocyte cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in an adipogenic medium such as a culture medium which comprises IBMX (3-isobutyl-l-methylxanthine), Dexamethasone and insulin, thereby generating the adipocyte cells.
  • an adipogenic medium such as a culture medium which comprises IBMX (3-isobutyl-l-methylxanthine), Dexamethasone and insulin
  • the pericyte progenitor cells e.g., seeded at a concentration of 2 x 10 s cells/cm 2
  • the pericyte progenitor cells are grown for about 4 weeks in the presence of 0.5 mM IBMX (3-isobutyl-l-methylxanthine), 10 ⁇ g/ml Insulin, 10 '6 M Dexamethasone, and 0.1 mM Indomethacin in DMEM/F12 medium (Biological Industries, Biet HaEmek, Israel) containing 10% FBS.
  • the adipocyte cells are characterized by accumulation of lipid-rich vacuoles which are positive for Oil red staining.
  • a method of generating chondrocyte cells comprising culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a chondrogenic culture medium such as a culture medium which comprises dexamethasone, ascorbic acid and TGF ⁇ 3, thereby generating the chondrocyte cells.
  • a chondrogenic culture medium such as a culture medium which comprises dexamethasone, ascorbic acid and TGF ⁇ 3, thereby generating the chondrocyte cells.
  • the pericyte progenitor cells (about 2xlO 5 cells) are centrifuged at 30Og for 5 minutes in 15 ml polypropylene falcon tubes to form a cell pellet.
  • the cells are grown in the presence of 10 ng/ml TGF ⁇ 3 (Peprotech) in DMEM medium for 9 weeks with media changes twice a week without disturbing the cell mass.
  • the pellets are cultured in medium containing 1 % serum in addition to high-glucose Dulbecco's modified Eagle's medium supplemented with 10 ⁇ 7 M dexamethasone, 50 ⁇ g/ml ascorbate-2-phosphate, 40 ⁇ g/ml L-proline, 100 ⁇ g/ml sodium pyruvate, 50 mg/ml ITS+Premix (Collaborative Biomedical: 6.25 ⁇ g/ml insulin, 6.25 ⁇ g/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid) and 10 ng/ml TGF- ⁇ 3.
  • medium containing 1 % serum in addition to high-glucose Dulbecco's modified Eagle's medium supplemented with 10 ⁇ 7 M dexamethasone, 50 ⁇ g/ml ascorbate-2-phosphate, 40 ⁇ g/ml
  • sub-confluent pericyte progenitor cell cultures are removed from the culture plates (without pre-collagenase treatment) as an intact layer, placed in suspension and fed with a medium containing alpha-MEM, supplemented with 15 % fetal bovine serum (FBS), 50 ⁇ g/ml ascorbic acid, 10 ⁇ 7 M and dexamethasone.
  • FBS fetal bovine serum
  • the chondrocyte cells are characterized by positive von-Kossa staining.
  • a method of generating myoblast cells is effected by culturing the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a culture medium which comprises horse serum, thereby generating the myoblast cells.
  • the myoblast cells are characterized by expression of markers such as MyoD and anti-smooth muscle myosin heavy chain.
  • the pericyte progenitor cells of some embodiments of the invention (5xl0 4 /cm 2 ) are incubated in M-199 supplemented with
  • the medium consisted of DMEM supplemented with 2% horse serum. Half of the medium was changed every 4 days for the following 7-10 days.
  • the pericyte progenitor cells of some embodiments of the invention can be cultured in the presence of MS5 stromal cells (which are cultured in u ⁇ coated plates in a MEM medium supplemented by 10% FBS, 1% penicillin- streptomycin). Pericytes are co-cultured for 8-10 days in proliferation medium: 78.5% DMEM high-glucose, 10% FBS, 10% horse serum
  • mice For myogenesis in vivo, eight- to 12-week old SCID-NOD mice are used. The mice are anaesthetized by inhalation of isofluorane/02. Cardiotoxin (1.5 ⁇ g/ ⁇ l) is injected into the muscle one to 3 hours prior to cell transplantation. The pericyte progenitor cells of some embodiments of the invention are suspended in PBS (e.g., 35 ⁇ l) and then injected into the injured muscle. Mice are sacrificed 3 weeks after transplantation and muscle is harvested for immunohistochemistry analysis, essentially as described in U.S. Patent Publication No. 2007/0264239, which is fully incorporated herein by reference. "
  • the pericyte progenitor cells of some embodiments of the invention are injected into the gastrocnemius muscle of female NOD-SCID mice (6 to 8 weeks old) which had been injured by intramuscular injection of 15 ⁇ l of 50 ⁇ M cardiotoxin (Sigma) 2 hours earlier. Eighteen to twenty days after transplantation, the gastrocnemius muscles are harvested, flash frozen in liquid nitrogen-cooled 2-methylbutane, and serially sectioned. Spectrin staining can be used to detect human cell derived myofibers, essentially as described in U.S. Patent Publication No. 2007/0264239, which is fully incorporated herein by reference.
  • the ability of the pericyte progenitor cells of some embodiments of the invention to recover cardiac function can be tested in an animal model essentially as described in U.S. Patent Publication No. 2007/0264239, which is fully incorporated herein by reference.
  • Myocardial infarction is induced in anesthetized nude rats via ligation of the left anterior descending coronary artery.
  • the pericyte progenitor cells of some embodiments of the invention in a PBS medium are immediately injected into the contracting wall bordering the infarct and into its center.
  • One, 2, 6, and 12 weeks later one population of rats is sacrificed and hearts are harvested, frozen and serially cryosectioned.
  • FISH fluorescent in situ hybridization
  • Anti- cardiac troponin I anti-atrial natriuretic peptide (ANP), anti-Nkx2.5, anti-.alpha.- myosin heavy chain ( ⁇ -MHC), anti-GATA-4, anti-connexin43 are used to investigate the acquisition of a myocardiac phenotype by the injected cells.
  • Capillary density in the heart cryosections is monitored after anti-vWF, anti-CD 144, anti-CD34 and anti-CD31 immunostaining, as is the expression of VEGF and VEGF receptor (KDR).
  • a method of generating smooth muscle cells in vivo comprising implanting the isolated pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention in a subject in need thereof, on in a tissue of a subject in need thereof, thereby generating the smooth muscle cells in vivo.
  • the isolated pericyte progenitor cells are mixed with an extracellular matrix such as Matrigel, collagen and the like.
  • a method of isolating an endothelial progenitor cell from embryoid bodies is effected by: (a) isolating CD105+, CD73+ and/or CD105+/CD73+ cells from the embryoid bodies, to thereby obtain CD105+, CD73+ and/or CD105+/CD73+ cells, and;
  • CD105+/CD73+ cells thereby isolating the endothelial progenitor cell from the pluripotent stem cells.
  • isolating endothelial progenitor cell is effected using pluripotent stem cells, which are induced to form embryoid bodies prior to isolation of the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • endothelial progenitor cell refers to an endothelial cells having the ability to further differentiate to additional non-endothelial cells.
  • endothelial cells are characterized by the expression of CD31, VE-
  • Cadherin Cadherin, UEA-I and by lack of expression of ⁇ SMA.
  • EBs from about day 7-26 are used.
  • Isolating the CD31+/Ulex europaeus lectin (UEA-l)+/VE-cadherin+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells can be performed using an antibody or antibodies which specifically bind CD31+, UEA-1+, and/or Ve-cadherin using any of the above described immuno-isolation methods.
  • CD31 [platelet/endothelial cell adhesion molecule, official symbol: PECAMl, also known as PECAM-I; FLJ58394] can be detected and/or isolated using specific antibodies such as CD31 antibody (ab32457; ABCAM), Clone MEM-05 (Abeam), Clone hcl/6 from AbD Serotec, or Clone JC70A (DAKO).
  • Ulex europaeus lectin is a lectin.
  • Non-limiting examples of antibodies which can specifically bind to cells expressing UEA-I include NB110-13922 (Novus Biologicals), ab50683 (ABCAM), U4754 (Sigma-Aldrich) or FITC-L9006 (Sigma- Aldrich).
  • VE-cadherin [cadherin 5, type 2 (vascular endothelium), official symbol: CDH5, also known as 7B4; CD144; FLJ17376; CDH5, Swiss Prot: P33151] can be detected and/or isolated using specific antibodies such as clone EPR3111Y (Catalogue No. 2465-1, Epitomics), Anti-Human CD144 (VE-Cadherin) PE (Catalogue No. 12-1449, eBioscience), VE Cadherin antibody (ab33168, ABCAM) and PE-conjugated mouse anti- human VE-Cadherin (Catalogue No. FAB9381P, R&D, Systems).
  • specific antibodies such as clone EPR3111Y (Catalogue No. 2465-1, Epitomics), Anti-Human CD144 (VE-Cadherin) PE (Catalogue No. 12-1449, eBioscience), VE Cadherin antibody (ab33168, ABCAM) and PE-conju
  • the method further comprising culturing the CD105+, CD73+ and/or CD105+/CD73+ cells for one or two passages prior to the isolating the CD31+/UEA-l+/Ve-cadherin+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells.
  • the endothelial cells isolated by the method of some embodiments of the invention have an expression marker signature of CD31+/Ulex europaeus lectin (UEA-l)+/VE-cadherin+.
  • the endothelial cells isolated by the method of some embodiments of the invention express vW Factor [Von Willebrand factor, VWF, also known as VWD; F8VWF; is a glycoprotein which functions as both an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system]; Flkl [VEGFR2, kinase insert domain receptor (a type III receptor tyrosine kinase), also known as CD309; VEGFR; KDR]; CD34; and/or FIt-I [VEGFRl, fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor), also known as FLT].
  • VW Factor Von Willebrand factor, VWF, also known as VWD
  • F8VWF is a glycoprotein which functions as both an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system
  • Flkl Flk
  • the endothelial progenitor cells isolated by the method of some embodiments of the invention have an expression marker signature of CD31+/Ulex europaeus lectin (UEA-l)+/VE-cadherin+/vW
  • the endothelial progenitor cells can be cultured under non-differentiating conditions such as on a matrix (such as fibronectin extracellular matrix or gelatin) in the presence of a culture medium such as endothelial culture medium such as M-199
  • the endothelial cells can be passaged every about 5-10 days as described in the Examples section which follows and in Daylon et al., 2009 (Nature Biotechnology, advanced online publication, which is fully incorporated herein by reference).
  • the endothelial progenitor cells can differentiatiate into cells which express ⁇ SMA (See for example, Figure 7F, Example 2 of the Examples section which follows).
  • an isolated population of endothelial progenitor cells generated according to the method of some embodiments of the invention.
  • the isolated population of endothelial progenitor cells comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100% of endothelial progenitor cells having the CD31+/Ulex europaeus lectin (UEA- 1)+/VE- cadherin+ signature.
  • the isolated population of endothelial progenitor cells comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100% of endothelial progenitor cells having the CD31+/Ulex europaeus lectin (UEA-1)+/VE- cadherin+/vW Factor+/Flkl+/CD34+/Flt-1+ signature.
  • UVA-1 CD31+/Ulex europaeus lectin
  • the isolated population of endothelial progenitor cells comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, e.g., 100% of endothelial progenitor cells having the CD31+/Ulex europaeus lectin (UEA-1)+/VE- cadherin+/vW Factor+/Flkl+/CD34+/Flt-l+/CD105+/CD73+/NG2-/PDGFR- ⁇ -/CD90- signature.
  • UVA-1 CD31+/Ulex europaeus lectin
  • pericyte and endothelial progenitor cells can be further expanded and used for various applications either as separated population of cells or as a combined population of cells in various ratios therebetween, such as 1:2, 1:1, 2:1, 1:3, 3:1 or pericyte/endothelial progenitor cells.
  • a method of co-derivation of pericyte and endothelial progenitor cells is effected by: (a) isolating CD105+, CD73+ and/or CD105+/CD73+ cells from embryoid bodies, to thereby obtain CD105+, CD73+ and/or CD105+/CD73+ cells; (b) isolating a CD31+/UEA-l+/Ve-cadherin+ cells from the CD105+, CD73+ and/or CD105+/CD73+ cells, to thereby isolate the endothelial progenitor cells; (c) isolating CD31- cells from the CD105+, CD73+ and/or CD105+/CD73+ cells, to thereby isolate the pericyte progenitor cells; thereby co-derivation of the pericyte and endothelial progenitor cells.
  • the method uses pluripotent stem cells, from which the embryoid, bodies are generated prior to isolating CD105+, CD73+ and/or CD105+/CD73+ cells from the embryoid bodies.
  • the CD105+, CD73+ and/or CD105+/CD73+ cells are isolated from EBs of day about 7-26 of EBs differentiation.
  • steps (b) and (c) are performed on two separated culture dishes or flasks, in order to isolate the two different populations of pericyte progenitors and endothelial progenitors.
  • the method further comprising: culturing the CD31+/UEA-l+/Ve-cadherin+ cells, to thereby expand the endothelial progenitor cells.
  • isolating and/or culturing the CD31+/UEA-l+/Ve-cadherin+ cells results in an isolated population of endothelial progenitor cells comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • endothelial progenitor cells as described above (e.g., having the CD31+/Ulex europaeus lectin (UEA- l)+/VE-cadherin+ signature).
  • the method further comprising culturing the CD31- cells, to thereby expand the pericyte progenitor cells.
  • pericyte progenitor cells isolated by co-derivation can be cultured on low-adhesive culture plates/flasks which can be non-coated.
  • isolating and/or culturing the CD31- cells results in an isolated population of pericyte progenitor cells comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • pericyte progenitor cells as described above (e.g., having the CD105+/CD73+/CD31-/ ⁇ SMA- signature).
  • teachings of the invention can be used to generate two populations of cells, wherein one population comprises pericyte progenitor cells and the other population comprises endothelial progenitor cells.
  • an isolated population of pericyte progenitor cells generated by co-derivation as described above.
  • an isolated population of endothelial progenitor cells generated by co-derivation as described above.
  • an isolated population of pericyte and endothelial progenitor cells generated according to the method of some embodiments of the invention.
  • a cell culture comprising a culture medium and the isolated endothelial progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention.
  • the medium can be any liquid medium suitable for culturing the endothelial progenitor cells.
  • Non-limiting examples of such a medium include, but are not limited i
  • endothelial cell medium M-199 medium supplemented with FBS and ECGS and heparin.
  • a cell culture comprising a culture medium and the isolated endothelial and pericyte progenitor cell of some embodiments of the invention, or the isolated population of cells of some embodiments of the invention.
  • the medium can be any liquid medium suitable for culturing the endothelial and pericyte progenitor cells.
  • Non-limiting examples of such a medium include, but are not limited to the endothelial cell medium M-199 medium supplemented with FBS and ECGS and heparin.
  • the isolated pericyte and/or endothelial progenitor cells of some embodiments of the invention can be used for repairing and/or regenerating vascular tissues.
  • a method of treating a pathology requiring vascular tissue regeneration and/or repair is provided.
  • the method is effected by administering to a subject having the pathology the isolated pericyte progenitor cell of some embodiments of the invention, the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, the isolated population of endothelial progenitor cells of some embodiments of the invention, or a pharmaceutical composition comprising same, thereby treating a pathology requiring vascular tissue regeneration and/or repair.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder or condition
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • subject includes mammals, preferably human beings at any age which suffer from the pathology.
  • Non-limiting examples of diseases/pathologies/conditions which require vascular tissue regeneration and/or repair include ischemia, diabetes, diabetic microangiopathy, peripheral arterial disease, cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S.
  • Administration of the cells of some embodiments of the invention can be effected using any suitable route such as intravenous, intra cardiac, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal.
  • the cells of some embodiments of the invention can be derived from either autologous sources such as self skin cells which are induced to become iPSCs and which are further used by the method of some embodiments of the invention or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.
  • Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
  • microcapsules Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479- 83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha- cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
  • microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 ⁇ m.
  • HEMA 2-hydroxyethyl methylacrylate
  • MAA methacrylic acid
  • MMA methyl methacrylate
  • Such microcapsules can be further encapsulated with additional 2-5 ⁇ m ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).
  • microcapsules are based on alginate, a marine polysaccharide (Sambanis,
  • microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
  • nanoporous biocapsules with well-controlled pore size as small as 7 run, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T.A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).
  • the isolated pericyte progenitor cell of some embodiments of the invention, the isolated pericyte progenitor cell of some embodiments of the invention, the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, and/or the isolated population of endothelial progenitor cells of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the isolated pericyte progenitor cell of some embodiments of the invention, the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, and/or the isolated population of endothelial progenitor cells of some embodiments of the invention accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially trans ⁇ asal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, vascular tissue, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions ill oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (the isolated population of pericyte progenitor cells of some embodiments of the invention, the cell culture of some embodiments of the invention, the isolated population of pericyte and endothelial progenitor cells of some embodiments of the invention, the isolated population of endothelial progenitor cells of some embodiments of the invention ) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a pathology requiring vascular tissue regeneration and/or repair) or prolong the survival of the subject being treated.
  • a disorder e.g., a pathology requiring vascular tissue regeneration and/or repair
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • teachings of the invention are of both clinical, basic research and industrial importance.
  • the methods of the invention offer scalable culture systems for clinical and industrial purposes.
  • the isolated pericyte progenitor cells of some embodiments of the invention and/or cells differentiated therefrom can be used to stabilize blood vessels [see e.g., Darland, et al., Angiogenesis (2001) 4, 11-20; Carmeliet, Nat Med (2003) 9, 653-660; Jain, Nat Med (2003) 9, 685-693; Sieminski, et al., Tissue Eng (2002) 8, 1057-1069; Koike, et al., Nature (2004) 428, 138-139; Black, et al., FASEB J (1998) 12, 1331- 1340; Shinoka, et al., Artif Organs (2002) 26, 402-406)];
  • the isolated endothelial cells of some embodiments of the invention can induce the differentiation of undifferentiated mesenchymal cells into smooth muscle cells [See e.g., Flamme, et al., J Cell Physiol (1997) 173, 206-210; Rossant, et al., Curr Opin Genet De
  • the pericyte and/of endothelial progenitor cells of some embodiments of the invention can be used to screen for drugs having an effect on tube formation and/or regeneration of vascular tissue.
  • hESCs or iPSCs Normal pluripotent stem cells
  • disease-related pluripotent stem cells e.g., cells derived from a subject having a known disease, such as a genetic disease, a vascular disease, cancer, diabetes, arthrosclerosis, metabolic disease
  • pericyte progenitor cells e.g., a vascular disease, cancer, diabetes, arthrosclerosis, metabolic disease
  • endothelial progenitor cells e.g., endothelial progenitor cells which are further used to generate vascular tubes in vitro, e.g., using a matrix such as Matrigel (BD Biosciences).
  • a matrix such as Matrigel (BD Biosciences).
  • EPCs are seeded on Matrigel coated slides or 24 well dishes in M-199 medium supplemented with 20% FBS without ECGS and heparin. Tube formation is documented 2, 12 and 48 hours post seeding.
  • Various drugs can be added before seeding, at pre-determined time points after seeding (i.e., during tube formation), and/or after the tubes are formed. The effect of the drugs on the kinetic of tube formation, and/or on tube structure can be detected by monitoring the structure/morphology of the formed micro tubes. Drug molecules capable of repairing tube formation, and/or kinetic of tube formation can be selected.
  • Pericyte progenitor cells, and/or endothelial progenitor cells which are generated from normal or diseased pluripotent stem cells can be mixed with a matrix (e.g., Matrigel assay) and further injected to the back or lateral flank of NOD/SCID mice (e.g., 6-8 old).
  • Drugs can be injected to the implanted animals at various time points before, during or following implantation, and the effect of the drug on tube formation can be evaluation by various histological and/or immunohistochemical assays.
  • Pericyte progenitor cells and/or endothelial progenitor cells which are generated from pluripotent stem cells can be used to generate vasculature ex vivo and/or in vivo. These cells can be used to establish clinical transplantation protocols for treatment of vascular disorders.
  • scaffolds or tissue grafts e.g., biological, synthetic, biodegradable and the like
  • pericyte progenitor cells e.g., hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hematoma, hem
  • scaffolds or tissue grafts e.g., biological, synthetic, biodegradable and the like
  • the pericyte progenitor cells, and/or endothelial progenitor cells can be seeded with the pericyte progenitor cells, and/or endothelial progenitor cells and further implanted in an animal model/subject in need thereof to test the ability of the vascular cells to repair, regenerate and/or form a vascular tissue.
  • the cells can be cultured ex vivo prior to being implanted in a subject.
  • the scaffold or tissue graft can be implanted to the subject/animal model without the cells and the cells can then be administered to the site of implantation or in vicinity thereto after graft implantation.
  • tissue grafts which can be seeded with the pericyte progenitor cells, and/or endothelial progenitor cells prior to, concomitant with, or following implantation of the tissue grafts: cardiac muscle tissue graft (for generation of vasculature for cardiac muscle), pancreatic tissue graft (islets of Langerhans), liver graft, bone graft, cartilage graft, skeletal muscle graft, and lung graft.
  • cardiac muscle tissue graft for generation of vasculature for cardiac muscle
  • pancreatic tissue graft islets of Langerhans
  • liver graft for generation of vasculature for cardiac muscle
  • pancreatic tissue graft islets of Langerhans
  • liver graft for generation of vasculature for cardiac muscle
  • bone graft for generation of vasculature for cardiac muscle
  • cartilage graft cartilage graft
  • skeletal muscle graft skeletal muscle graft
  • pericyte progenitor cells, and/or endothelial progenitor cells of some embodiments of the invention for understanding the pathogenesis of various diseases - Normal and diseased pluripotent stem cells (as described above) can be used to generate pericyte progenitor cells, and/or endothelial progenitor cells which are used in various models to understand the pathogenesis of a disease.
  • tube formation can be compared between pericyte progenitor cells, and/or endothelial progenitor cells which are generated from iPSCs derived from a diabetic subject and from iPSCs of a healthy individual.
  • pericyte progenitor cells, and/or endothelial progenitor cells of some embodiments of the invention can be used for better understanding developmental processes of vasculogenesis as well as identification of interactions between the blood vessel cellular compartments.
  • pericyte progenitor cells and/or endothelial progenitor cells of some embodiments of the invention: tissue reconstruction, reconstructive surgery,
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
  • the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term "treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subco ⁇ ibinatiori or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
  • PSCs Human pluripotent stem cell (PSCs) culture and differentiation - Human ESC H9.2 (passages 29+36-60, i.e., the H9 cell line was cloned on passage 29, and the H9.2 cell line clone was used at passages 36-60), 16 (passages 50-71) or human foreskin fibroblasts derived iPSC (Germanguz et al, J Cell MoI Med. 2009 Dec 11.
  • clones C2 and C3 (passages 17-38) and hair follicle keratinocyte derived iPSC clone KTR13 (passages 30-49) were grown on mitomycin C (1 mg/ml) inactivated mouse embryonic fibroblasts (MEF) in ESC culture medium consisting of: advanced DMEM/F12 (Biological Industries, Biet HaEmek, Israel) supplemented with 20% knockout serum replacement (GIBCOTM KnockoutTM Serum Replacement ), Ix nonessential amino acids (Gibco), Ix 1-glutamine (Invitrogen), Ix ⁇ -mercaptoethanol (Gibco) and 4 ng/ml FGF-2 (R&D systems) without antibiotics.
  • mitomycin C (1 mg/ml) inactivated mouse embryonic fibroblasts (MEF) in ESC culture medium consisting of: advanced DMEM/F12 (Biological Industries, Biet HaEmek, Israel) supplemented with 20% knockout serum replacement (GIBCO
  • Embryoid bodies formation and differentiation To induce spontaneous embryoid bodies (EBs) formation and differentiation, PSCs were removed from MEF feeder by 0.2% Collagenase IV (Gibco Invitrogen Corporation, Grand Island NY, USA) and were suspended in low attachment culture dishes in differentiating medium consisting of DMEM, 20% FBS, Ix non-essential amino acids, Ix 1-glutamine, Ix ⁇ - mercaptoethanol without antibiotics. Culture medium was changed every 3 days.
  • EBs dissociation protocols To optimize the dissociation step for obtaining single cell suspensions, three methods of dissociating EBs were tested. These include incubation of EBs for 20 minutes at 37°C on shaker with either (1) 0.5% Trypsin/EDTA (Sigma), (2) non-enzymatic solution (Sigma, catalogue No. C5914) or (3) 1 mg/ml collagenase B and 150 U/ml DNAse I (Catalogue No. 2139, Roche), followed by addition of Trypsin-EDTA (0.05%) for another 5 minutes.
  • Trypsin/EDTA Sigma
  • non-enzymatic solution Sigma, catalogue No. C5914
  • 1 mg/ml collagenase B and 150 U/ml DNAse I Catalogue No. 2139, Roche
  • the dissociated EBs were then passed several times through a 20-Gauge needle and filtered through PBS/0.5% FBS pre-washed 0.45 cell-strainers (BD Biosciences). Cell viability was determined by Trypan Blue assay, and the percentages of live CD31 + endothelial progenitor cells (EPC) or CD105+ cells were analyzed by flow cytometry upon dissociation with Colagenase B.
  • EPC endothelial progenitor cells
  • CD105+ cells were analyzed by flow cytometry upon dissociation with Colagenase B.
  • Pericyte and endothelial progenitor cell isolation were made from differentiated hPSC (EBs) by treatment with 1 mg/ml collagenase B and 150 U/ml DNAse I as detailed above.
  • CD 105+ cells were isolated from differentiated hPSC (EBs) by using MACS MicroBeads and MACS columns (Miltenyi Biotec, anti-human CD105 MicroBeads, Catalogue No. 130-051- 201, anti-human CD31 MicroBeads Catalogue No.
  • CD31+CD105+ endothelial cells were sorted/isolated from mixed culture of CD105+ isolated cells at passage 1-2 by magnetic separation using anti-CD31 MicroBeads conjugated antibodies or by sorting using FITC or PE-conjugated anti-human CD31 antibodies (purity of CD31 + >97%).
  • UEA-I based isolation was performed by cell sorting using UEA-I-FITC which resulted in CD105+/UEA-1+ endothelial cells and CD105+/UEA-1- pericyte progenitor cells.
  • Isolated CD31+CD105+ endothelial cells were further expanded.
  • PSC-derived CD31+/CD105+ EPCs from second isolation were cultured in coated fibronectin or gelatin plates in a medium such as EBM-2 (Catalogue No. CC-3156, Lonza, Walkerville, MD, USA) or EC M-199 growth media both supplemented with 10 ⁇ M SB431542 (Catalogue No. 1614, Tocris Biosciences, Bristol, UK) for expansion of endothelial cells as described (Daylon J., et al., 2010, Nat Biotechnol 28: 161-166).
  • CD105+CD31- pericytes from second isolation step (which used CD31 antibodies) were further expanded in EC M-199 growth media in uncoated plates.
  • CD105+/CD31- and CD105+/CD31+ cells obtained by sorting ofCD105+ cells - CD105+ isolated/sorted mixed culture of EPCs and pericytes; CD31+/CD105+ isolated/sotted endothelial progenitor cells (EPCs); or CD105+/CD31- isolated/sorted pericyte progenitor cells were seeded on human fibronectin (Millipore, Billerica, MA) coated culture dishes and cultured in endothelial cell (EC) M-199 media (Catalogue No.
  • Isolated cells of CD105+/CD31- obtained by expansion of CD105+ cells in culture -
  • the mixed population CD105+/CD31+ and CD105+/CD31- cells were co-cultured in EC M-199 growth media.
  • CD105+CD31- dominated the culture within 1- 2 passages, in accordance with the initial percentage of isolated CD105+CD31- within dissociated EBs.
  • CD90 clone EPR3133, Catalogue No. 2695-1
  • MHC class I clone EPR1394Y, Catalogue No. 2307-1
  • anti-Tie-2 (1:100, R&D Systems
  • mouse anti-human ⁇ -SMA DAKO, clone, 1A4, Catalogue No. M0851,
  • smooth muscle myosin heavy chain or CD31 Dako, 1:50
  • mouse anti-human CXCR4 clone 12G5, R&D Systems, 1:300.
  • FITC-conjugated Ulex europaeus lectin was also used as an endothelial cell marker (Sigma, 1:200).
  • the secondary conjugated antibodies included: Alexa-488 conjugated donkey anti-mouse, Alexa-488 conjugated donkey anti-rabbit, Alexa-488 conjugated donkey anti-goat (1:100, Invitrogen), Alexa-488 conjugated goat anti-rabbit, and Cy-3 conjugated donkey anti- mouse (1:100, Jackson).
  • Labeled DiI-Ac-LDL (5 ⁇ g/ml) was used for identification of endothelial cells according to the manufacturer instructions (Biomedical Technologies Inc).
  • CM-DiI labeling was performed according to the manufacturer instructions (Molecular probes). Slides were mounted in mounting medium (Catalogue No., S3023, Dako) and observed on an epifluorescence microscope (Ziess). Adherent cultures were viewed by Axiovert 200 equipped with AxioCam MRm camera.
  • Nuclei labeling with DAPI 40, 6-diamino-2-phenylindole dihydrochloride, Molecular Probes, 1:1000
  • DAPI 6-diamino-2-phenylindole dihydrochloride
  • Molecular Probes 1:1000
  • an isotype- matched negative control or irrelevant isotype-matched antibody e.g. mouse anti- human CD45, Dako
  • FAB9381P, R&D, Systems isotype control antibodies included PE-conjugated mouse IgG2B (Catalogue No. IC0041P, R&D, Systems), FITC-co ⁇ jugated mouse IgG l ⁇ (Catalogue No. BD
  • endothelial cells either human umbilical vein endothelial cells (HUVEC) or PSC-derived EC and/or PSC-derived pericytes were removed from culture dishes by 0.05% trypsin, washed and re-suspended in 250 ⁇ l phenol-red free Matrigel (BD Biosciences), either alone (3-5xl0 5 EC, 6-8xl0 5 pericytes) or mixed. Matrigel mixture was then injected to the back or lateral flank of 6- 8 old NOD/SCID mice. Implants were removed on the indicted days and fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and Eosin or further immunolabeled. Empty Matrigel served as control implants.
  • HUVEC human umbilical vein endothelial cells
  • PSC-derived EC and/or PSC-derived pericytes were removed from culture dishes by 0.05% trypsin, washed and re-suspended in 250
  • Adipogenic differentiation and Oil Red O staining - Pericytes were seeded (2 x 10 5 cells/cm 2 ) on culture dish, in the presence of 0.5 mM IBMX, 10 ⁇ g/ml Insulin, 10 '6 M Dexamethasone, and 0.1 mM Indomethacin in DMEM/F12 medium (Biological Industries, Biet HaEmek, Israel) containing 10% FBS for 4 weeks, with media changes twice a week.
  • Adipogenic differentiation was assessed by accumulation of lipid-rich vacuoles within the cells after Oil Red O staining as follows: cells were rinsed once with PBS, fixed with 4% Paraformaldehyde (PFA) for 20 minutes, rinsed again and stained with Oil Red O solution for 10 min in room temperature. Staining solution was removed and the cells were washed 5 times with water.
  • PFA Paraformaldehyde
  • Osteogenic differentiation in vitro - Pericytes were seeded at low density (2xlO 4 - 3xlO 4 cells/cm 2 ) on culture dish, in the presence of 10 mM ⁇ -glycerol-phosphate and 0.1 ⁇ M Dexamethasone (Catalogue No. D4902, Sigma- Aldrich) in GMEM BHK-21 medium (Catalogue No. 21710, Gibco) containing 10 % FBS for 4 weeks, with media changes twice a week.
  • Cell cultures were assayed for mineral content by Alizarin red staining as follows: treated cells were rinsed once with PBS, fixed with 4 % PFA for 20 minutes, rinsed again and stained with 2 % Alizarin red solution for 15 minutes in room temperature. Staining solution was removed and the cells were washed several times with water.
  • osteogenesis in vivo pericytes were cultured in osteogenic differentiation medium (1OmM ⁇ -glycerol -phosphate and 0.1 ⁇ M Dexamethasone (Catalogue No. D4902, Sigma-Aldrich) in GMEM BHK-21 medium (Catalogue, No. 21710, Gibco) for 3 or 14 days.
  • Treated cells were then removed from culture dishes and mixed with 250 ⁇ l Matrigel ((BD Biosciences) at a concentration of 3xl0 5 -10 6 cells/Matrigel implant of 250 ⁇ l, which was injected subcutaneously into immunodeficient 10-12 weeks old NOD/SCID mice. Implants were harvested after 1-2 weeks, fixed in 4% formaldehyde, embedded in paraffin, sectioned and stained with H&E or 2% Alizarin red solution for 15 min in room temperature.
  • Myogenic differentiation - PSC-derived pericytes (5xlO 4 /cm 2 ) were incubated in M-199 supplemented with 20% FBS for further growth.
  • Differentiation medium consisted of DMEM supplemented with 2% horse serum. Half of the medium was changed every 4 days for the following 7-10 days.
  • Fixed cells 4% paraformaldehyde
  • anti-smooth muscle myosin heavy chain (clone, SMMS-I Catalogue No. M3558, Dako) and anti-MyoD (clone 5.8A, Catalogue No. sc-32758, Santa-Cruz) for evaluation of myotubes formation.
  • RT-PCR - Total RNAs were isolated using Trizol/Tri-reagent (Invitrogen) and reverse transcribed reverse transcribed by the iScriptTM cDNA synthesis kit (BIO- RAD). RT-PCR was performed by DreamTaqTM Green Master Mix (Fermentas, Ontario, Canada). The sequences of oligonucleotide primers used for PCR are listed in Table 1 below.
  • Table 1 Provided are the primers used for RT-PCR along with their sequence identifiers (SEQ ID NO:) and gene name.
  • EBs embryoid bodies
  • PSC human pluripotent cells
  • the cell lines used were as follows: for human embryonic stem cells the H9.2 and 16 cell lines were used; for induced pluripotent stem cells (iPSCs) the C3 and KTR13 were used.
  • iPSCs induced pluripotent stem cells
  • EBs taken at any day from day 1-26 were dissociated by treatment with Collagenase B and DNAse I in order to prevent cell clumping and enable better dissociation of the EBs.
  • CD105+ cells Characterization of CD105+ cells in EBs at various differentiation stages - Expression analysis of EBs at different days of differentiation demonstrated that CD 105 expression was upregulated between days 4-26 in culture ( Figure IA). Flow cytometry analysis revealed that while CD105+ cells constitute only 0.08% of the total EBs cells at day 1 of EBs differentiation, the fraction of CD105+ cells increases to about 4.1% (which includes the 3.5% of CD105+/CD31- and the 0.6% of CD105+/CD31+) of the total EBs cell population at day 26.
  • CD105+ cells there were two distinguished cell subsets: until day 14 the majority of CD105 + cells were composed of CD105+CD31+ endothelial cells (i.e., 3% on day 14) ( Figures 2A-P and Figures 3A-D) with a small subset of non-endothelial CD105+CD31- population (i.e., 2 %) ( Figures 2A-J and Figures 3A-D). From day 17 onward the percentage of CD105+CD31+ endothelial cells as well as CD31 gene expression declined progressively coinciding with an increase in the percentage of CD105+CD31- non-endothelial subset ( Figures 2K-P and Figures 3A-D).
  • CD105+ positive and aSMA- (negative) -
  • CD3I- negative and aSMA- (negative) -
  • EC endothelial cell
  • 3 types of colonies could be identified within 7-10 days in culture: (1). CD31 + EC (endothelial cell) colonies; (2). Mixed CD31+ endothelial and CD31- ⁇ SMA+ cells with rare subset expressing both markers, and; (3). Non-ECs, of which the majority of cells were CD31 " ⁇ SMA " multilayered cells with a small subset of SMC ( Figures 7A-I).
  • the first two populations were previously described in various differentiation models of human ESC including spontaneously differentiating EBs (Levenberg S., et al., 2002, PNAS 99: 4391-4396), maturation on murine feeders (Wang Z., et al., 2007, Nat Biotechnol 25: 317-318;
  • CD105+/CD31-/ ⁇ xSMA- cells Domination of the CD105+/CD31-/ ⁇ xSMA- cells in culture - Within 2-3 passages in culture the CD105+/CD31- subset dominated the mixed PSC-derived vasculogenic cultures exhibiting loosen cell-cell contacts in a multilayered hill and valley morphology Figures 7G-I, a cell morphology which was previously described in pericyte cultures from animal or human fetal or adult source (Majack R. 1987, The Journal of Cell Biology 105: 465-471; Crisan M., et al., 2008, Cell Stem Cells 3: 301- 313) and which is typical of cultured pericytes as well as smooth muscle cells.
  • CD105+CD31- colonies (determined by flow cytometry analysis) emerging from PSC-derived CD105+ cells were cloned by limiting dilution at first passage and gave rise to single cell-derived colonies with similar morphology (data not shown) at clonal efficiency of 5 ⁇ 1.1% for H9.2 and 6 ⁇ 2% for iPSC C3.
  • pericyte progenitor cells in culture up to 4200 fold increase within 7-8 passages - PSC-derived CD31
  • PDT population doubling time
  • After 7 weeks of culture pericytes gradually entered hypertrophic senescence (Figure 9). Contact inhibition was not observable and adherent spherical clusters of multilayered cells were detached from the plastic at high densities (data not shown).
  • CD105+CD31- cells did not express skeletal muscle cells markers (CD56 and E- cadherin), specific markers of endothelial cells (CD31, vW Factor, VE-Cadherin), hematopoietic markers (CD45, CD14, CD38) cells, or hemangioblast cell markers (CD34, CD133, Flkl, FIt-I) ( Figures 10A-P, Table 2 hereinbelow, and data not shown). This was confirmed by RT-PCR at early and late passages of cultured pericytes ( Figure 11 and data not shown).
  • cultured pericytes were positive for identified perivascular markers including CD 146 ( Figures 10K- L), NG2 ( Figures 12C-D), Calponin ( Figures 11 and 12A-B) and PDGFR- ⁇ ( Figures 12 A-B), negative for the endothelial specific marker CD31 ( Figures 10M-N), as well as for hematopoietic cell markers such as CD45 (data not shown) CD14 monocytic marker ( Figures 10O-P).
  • CD 146 Figures 10K- L
  • NG2 Figures 12C-D
  • Calponin Figures 11 and 12A-B
  • PDGFR- ⁇ Figures 12 A-B
  • CD31 Figures 10M-N
  • CD45 data not shown
  • CD14 monocytic marker Figures 10O-P
  • pluripotent stem cells PSC derived CD105+/CD31- cells demonstrated positive immuno-labeling for recognized pericytic markers including NG2, PDGFR- ⁇ , Calponin, CD90, Tie-1 and Tie-2 ( Figures 12A-F, Figures 14A-B, Figures 29 A-F, Figure 3OC and Table 2 below).
  • PSC pluripotent stem cells
  • Table 2 Provided are the expression patterns of the isolated and cultured pericyte progenitor cells and endothelial progenitor cells (EPCs) according to the method of some embodiments of the invention from any passage beginning from passage 1 to senescence (passage 5 for ECs and passage 8 for pericytes).
  • EPCs endothelial progenitor cells
  • MSC Mesenchymal stem cells
  • Vasculogenic potential of pericytes was initially evaluated in vitro. Although tube formation on Matrigel is a common feature of endothelial cells, it has been previously demonstrated that fetal tissue derived- ⁇ SMA positive pericytes can actively create tubular networks (Bagley RG, et al., Cancer Res. 2005 Nov l;65(21):9741-50).
  • the PSC-derived pericytes generated according to the method of some embodiments of the invention formed short, intricate tubular structures on Matrigel within 2 hours (Figure 15A) with sprouting clusters in between ( Figure 31A), while HUVEC (data not shown) or PSC-derived CD31 + endothelial cells re-arranged in linear, asymmetric tube networks that regenerated within 12 hours ( Figure 15B).
  • a characteristic feature of pericytes in developing and adult tissue is their ability to form cell-cell contact with endothelial cells to form a vascular network.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • the isolated pericytes and endothelial cells express human MHC class I -
  • mesenchymal tissues including fat, cartilage, myogenic, and bone
  • short-term and long-term cultured pericytes were used from passages 1-8, wherein at passage 9 cells entered senescence.
  • Osteogenic differentiation in vivo - PSC-derived pericytes were cultured in osteogenic medium, removed after 3 or 14 days of osteogenic stimulation, mixed with Matrigel and implanted subcutaneously into NOD/SCID immune deficient mice for evaluation of osteogenesis by ectopic model in vivo.
  • Hematoxylin and Eosin staining revealed appearance of mineral deposits in vivo already after 3 days of stimulation in osteogenic medium (Figure 19C).
  • Increased amount of deposits within implant is seen when PSC-derived pericytes were stimulated for 14 days (Figure 19E).
  • the amount of calcium was noticeably increased with longer cultivation period (Figure 19F) compared to short stimulation of PSC-derived pericytes ( Figure 19D) in vitro as detected by alizarin red staining.
  • PSC-derived pericytes Differentiation to adipocytes - Adipogneic potential of PSC-derived pericytes was tested in vitro by cultivation in adipogenic medium for 4 weeks.
  • the majority of cultured PSC-derived pericytes (about 90%-95%) contained lipid vesicles positive for Oil red staining (H9.2 deriyed-pericytes, Figure 2OA; iPSC C3-derived pericytes, Figure 20B).
  • PSC-derived pericytes maintained their adipogenic potential in vitro throughout the whole culture period between passages 2-9 (data not shown).
  • Isolated cells at passage 0 were composed of two subsets: CD31+CD105+ or CD31+CD73+ endothelial cells and CD105+CD31- or CD73+CD31- pericytes ( Figure 23B, Figures 24A-C, Figures 2A-P and Figures 39A-F).
  • a second isolation step was preformed for separation of endothelial cells from pericytes ( Figures 26D and 26E) for further expansion of both populations from passage 1.
  • Antibodies against CD31 or UEA-I-FITC lectin were used for isolation of endothelial cell population (CD31+UEA-1+CD105+ or CD31+UEA- 1+CD73+) for further cultivation (positive fraction, Figure 23D and Figure 26C).
  • Negative cell fraction CD105+CD73+CD31- consisted of pericytes at passage 1 post second isolation step (negative fraction, Figure 23E and Figure 26B).
  • PSC-derived EPCs (CD31+CD105+ or CD31+CD73+), which were isolated based on expression of CD 105 or CD73, were further cultivated after a second isolation step at passage 1 up to passage 5 in human fibronectin-coated or gelatin coated culture dishes in EC M-199 growth medium or in EBM-2 medium supplemented with 10 ⁇ M TGF- ⁇ inhibitor SB431542 (Tocris).
  • Expanded endothelial cells exhibited characteristics of adult tissue counterparts in vitro and in vivo expressing: VEGFRl ( Figure 21D), VEGFR2 (Figure 21F), CD31 ( Figure 21H), UEA-I ( Figure 21J), vW Factor (Figure 26C) and VE-Cadherin ( Figure 26E).
  • CD105+ PSC-derived endothelial cells formed tube networks when seeded on Matrigel (Figure 27A), which were positive for UEA-I (green, Figure 27B) or CD31 (red, Figure 27C). Cobbelstone forming cells were seen in culture dishes until passage 4-5 ( Figure 27D). Expanded PSC-derived endothelial cell maintained CD31 as well as CD 105 expression throughout the whole culture period ( Figure 27E).
  • the present inventors have uncovered novel methods of isolating pericytes from pluripotent stem cells by developing a unique approach to trace the potential emergence of pericytes alongside with development of endothelial and SMC based on common (CD105, ⁇ -SMA) and specific (CD31, VE-Cadherin, UEA-I) vascular cell markers.
  • the inventors have identified a novel population of cells positive for recognized markers of pericyte, including NG2, and PDGFR- ⁇ , and CD 146 along with other markers of mesenchymal stem cells, but negative for ⁇ -SMA.
  • the unique expression pattern of the isolated population of cells implies that these cells are more primitive ancestors within pericyte hierarchy.
  • cultured pericytes exhibited angiogenic characteristics in vitro and in vivo and maintained the ability to differentiate into chondrogenic, myogenic and adipogenic cell lineages upon sustained expansion (e.g., even following 7-9 passages in culture).
  • PSC-derived pericytes exhibited highly efficient and rapid osteogenic response in vitro compared to their MSC counterparts as well as ectopic bone formation after implantation into NOD/SCID mice. These results demonstrate an efficient derivation of multipotent mesodermal pericytic-precursors from PSCs.
  • the invention provides a method for efficient co-derivation of the two blood vessel cellular components, pericytes and endothelial cells, from a single pluripotent cell source.
  • the method is based on two isolation steps, wherein a single specific cell surface marker is used in each step.
  • CD105 + cells are isolated from embryoid bodies by magnetic separation or fluorescent sorting.
  • the isolated CD105 + population is then cultured in endothelial cell media, which gives rise to two distinguished cell populations: CD105 + CD31 + UEA-l + endothelial cells and CD105 + CD31 " UEA-l " cells.
  • all cultured CD105 + cells are incubated with either UEA-I or CD31 + antibodies to distinguish between CD31 + endothelial population and CD31 " cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Vascular Medicine (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention porte sur des procédés d'isolement de cellules progénitrices de péricyte à partir de cellules souches pluripotentes telles que des cellules souches embryonnaires et des cellules souches pluripotentes induites, par l'isolement de cellules CD105+, CD73+ et/ou CD105+/CD73+ à partir de corps embryoïdes et facultativement par enrichissement des cellules avec des cellules CD31-. L'invention porte également sur des procédés d'isolement de cellules endothéliales et de co-dérivation de péricyte et de cellules progénitrices de cellules endothéliales à partir de corps embryoïdes, et sur des procédés de différentiation de celles-ci pour diverses applications thérapeutiques. En outre, l'invention porte sur une cellule progénitrice de péricyte isolée ayant une signature de marqueurs d'expression consistant en CD105+/CD73+CD31-/alpha SMA-/CD133-/Flk-1-.
EP10755000A 2009-08-17 2010-08-17 Cellules progénitrices de péricyte et leurs procédés de génération et d'utilisation Withdrawn EP2467470A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US27210309P 2009-08-17 2009-08-17
PCT/IL2010/000669 WO2011021194A2 (fr) 2009-08-17 2010-08-17 Cellules progénitrices de péricyte et leurs procédés de génération et d'utilisation

Publications (1)

Publication Number Publication Date
EP2467470A2 true EP2467470A2 (fr) 2012-06-27

Family

ID=43085902

Family Applications (1)

Application Number Title Priority Date Filing Date
EP10755000A Withdrawn EP2467470A2 (fr) 2009-08-17 2010-08-17 Cellules progénitrices de péricyte et leurs procédés de génération et d'utilisation

Country Status (3)

Country Link
US (2) US20120148546A1 (fr)
EP (1) EP2467470A2 (fr)
WO (1) WO2011021194A2 (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230145494A (ko) 2006-04-14 2023-10-17 아스텔라스 인스티튜트 포 리제너러티브 메디슨 혈관 콜로니 형성 세포
WO2010129775A1 (fr) 2009-05-06 2010-11-11 University Of Virginia Patent Foundation Lentille portative à éclairage autonome pour examen et photographie de la rétine, et procédé associé
US20120045202A1 (en) * 2010-08-17 2012-02-23 Xu Jiang High Speed Bi-Directional Transceiver, Circuits and Devices Therefor, and Method(s) of Using the Same
AU2011291537B2 (en) * 2010-08-19 2016-06-02 The Regents Of The University Of California Compositions comprising perivascular stem cells and Nell-1 protein
US9809798B2 (en) * 2011-03-11 2017-11-07 National University Of Singapore Pericyte progenitors from peripheral blood
JP2014529406A (ja) * 2011-08-29 2014-11-13 セーフガードセル テクノロジー インク． 血管周囲薬用細胞の単離および治療的使用
WO2014138793A1 (fr) * 2013-03-13 2014-09-18 The University Of Queensland Procédé d'isolement de cellules pour la thérapie et la prophylaxie
US9506037B2 (en) 2013-03-15 2016-11-29 The Johns Hopkins University Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix
EP2970902B1 (fr) * 2013-03-15 2019-11-20 The Johns Hopkins University Réseau vasculaire auto-organisé de cellules souches pluripotentes humaines dans une matrice synthétique
US9994825B2 (en) * 2013-03-15 2018-06-12 The Johns Hopkins University Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix
EP3003290B1 (fr) 2013-06-05 2021-03-10 AgeX Therapeutics, Inc. Compositions pour l'utilisation dans le traitement des plaies chez des espèces mammaliennes
US9868939B2 (en) 2013-06-12 2018-01-16 Wisconsin Alumni Research Foundation Generating vasculogenic cell populations
WO2015119642A1 (fr) 2014-02-10 2015-08-13 The Johns Hopkins University Amélioration du devenir endothélial de cellules souches pluripotentes humaines par une faible pression d'oxygène
US11078462B2 (en) * 2014-02-18 2021-08-03 ReCyte Therapeutics, Inc. Perivascular stromal cells from primate pluripotent stem cells
US10240127B2 (en) 2014-07-03 2019-03-26 ReCyte Therapeutics, Inc. Exosomes from clonal progenitor cells
WO2022196714A1 (fr) 2021-03-17 2022-09-22 アステラス製薬株式会社 Péricyte ayant un gène de facteur de croissance fibroblastique basique (bfgf) introduit dans celui-ci
JPWO2023286834A1 (fr) 2021-07-15 2023-01-19
JPWO2023286832A1 (fr) 2021-07-15 2023-01-19

Family Cites Families (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL154600B (nl) 1971-02-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen.
NL154598B (nl) 1970-11-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van laagmoleculire verbindingen en van eiwitten die deze verbindingen specifiek kunnen binden, alsmede testverpakking.
NL154599B (nl) 1970-12-28 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen, alsmede testverpakking.
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
NL171930C (nl) 1972-05-11 1983-06-01 Akzo Nv Werkwijze voor het aantonen en bepalen van haptenen, alsmede testverpakkingen.
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
US5843780A (en) 1995-01-20 1998-12-01 Wisconsin Alumni Research Foundation Primate embryonic stem cells
US6090622A (en) 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
US20040009589A1 (en) 2002-03-26 2004-01-15 Shulamit Levenberg Endothelial cells derived from human embryonic stem cells
US7354763B2 (en) 2002-04-16 2008-04-08 Technion Research & Development Foundation Ltd. Generating vascular smooth muscle cells in vitro from ES cells
US7247477B2 (en) 2002-04-16 2007-07-24 Technion Research & Development Foundation Ltd. Methods for the in-vitro identification, isolation and differentiation of vasculogenic progenitor cells
KR20060114366A (ko) 2004-02-09 2006-11-06 인디애나 유니버시티 리서치 앤드 테크놀로지 코포레이션 클론원성 내피 전구 세포의 분리, 팽창 및 용도
EP1799810A2 (fr) 2004-10-12 2007-06-27 Technion Research & Development Foundation Ltd. Cellules embryonnaires de primate isolee et procedes de generation et utilisation de telles cellules
US20060198827A1 (en) 2005-02-04 2006-09-07 Shulamit Levenberg Engineering vascularized muscle tissue
US20070264239A1 (en) 2006-05-10 2007-11-15 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Isolation of pericytes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHUNG YOUNG ET AL: "Human embryonic stem cell lines generated without embryo destruction", CELL STEM CELL, CELL PRESS, US, vol. 2, no. 2, 7 February 2008 (2008-02-07), pages 113 - 117, XP002604696, ISSN: 1934-5909, DOI: 10.1016/J.STEM.2007.12.013 *

Also Published As

Publication number Publication date
WO2011021194A2 (fr) 2011-02-24
US20170342384A1 (en) 2017-11-30
US20120148546A1 (en) 2012-06-14
WO2011021194A3 (fr) 2011-06-03

Similar Documents

Publication Publication Date Title
US20170342384A1 (en) Pericyte progenitor cells and methods of generating and using same
Teresa Conconi et al. Phenotype and differentiation potential of stromal populations obtained from various zones of human umbilical cord: an overview
Mosna et al. Human bone marrow and adipose tissue mesenchymal stem cells: a user's guide
Kerkis et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers
US8017389B2 (en) Enriched stem cell and progenitor cell populations, and methods of producing and using such populations
US9018005B2 (en) Method of deriving progenitor cell line
Jing et al. Stem cells for heart cell therapies
EP2268795B1 (fr) Procédé pour générer des cellules progénitrices cardiaques de primate pour un usage clinique à partir de cellules souches embryonnaires, et leurs applications
US20060171932A1 (en) Adipose derived stromal cells exhibiting characteristics of endothelial cells
Lee et al. Novel embryoid body–based method to derive mesenchymal stem cells from human embryonic stem cells
Liu et al. Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan
JP5265537B2 (ja) ヒト胚盤胞由来幹細胞に由来する多能性心臓前駆細胞の新規の集団
Gao et al. Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs
US8771677B2 (en) Colony-forming unit cell of human chorion and method to obtain and use thereof
Rady et al. Mesenchymal stem/progenitor cells: the prospect of human clinical translation
Liu et al. Generation, characterization, and potential therapeutic applications of cardiomyocytes from various stem cells
JP2023531975A (ja) 間葉系幹細胞を産生するための培地及び方法
US9598675B2 (en) Method for preparing mesenchymal stem cell-like cells and cardiomyocyte-like cells
Mahmood et al. Derivation of stromal (skeletal and mesenchymal) stem-like cells from human embryonic stem cells
Moon et al. Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells
Bettiol et al. Embryonic and adult stem cell-derived cardiomyocytes: lessons from in vitro models
IL178006A (en) Method for making high purity cardiomyocyte preparations suitable for regenerative medicine
Zhang et al. Isolation and biological characterization of muscle-derived stem cells from sheep skeletal muscle
Emanueli et al. In search of the best candidate for regeneration of ischemic tissues
WO2021227573A1 (fr) Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20120227

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20130304

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 5/073 20100101AFI20170629BHEP

Ipc: A61K 35/44 20150101ALI20170629BHEP

Ipc: A61K 35/545 20150101ALI20170629BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20170810

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20171221